Jeremy Vanhelst

Laurent Beghin

Alain Duhamel

Stefaan De Henauw

Dénes Molnar

German Vicente-Rodríguez

Yannis Manios

Kurt Widhalm

Mathilde Kersting

Angela A Polito

Jérémy Vanhelst

Laurent Béghin

Denes Molnar

Germán Vicente-Rodriguez

Jonatan R Ruiz

Luis A Moreno

Frédéric Gottrand

Relationship between school rhythm and physical activity in adolescents: the HELENA study

Keywords: Physical Activity, School rhythm, Health, Adolescents

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Physical inactivity levels are rising in many countries, and this increase has major implications for the prevalence of obesity throughout the European countries.

Physical inactivity is now identified as the fourth leading risk factor for global mortality [START_REF] Who | Global health risks: mortality and burden of disease attributable to selected major risks[END_REF]. To face this challenge, in 2006 the European member states adopted the European Charter on Counteracting Obesity as a part of the new European health policy (WHO, 2010). It includes the recommendation of 60 minutes of moderate-to-vigorous physical activity (MVPA) every day (WHO, 2010;[START_REF] Cale | Exercise recommendations for young people: An update[END_REF]. However, a large proportion of youth do not achieve this recommended level of MVPA [START_REF] Riddoch | Physical activity levels and patterns of 9-and 15-yr-old European children[END_REF][START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF]. In 2004, from 2185 adolescents assessed by the accelerometry, 18% of boys and 38% of girls did not achieve the PA recommendations [START_REF] Riddoch | Physical activity levels and patterns of 9-and 15-yr-old European children[END_REF]. In a population of 2200 European adolescents, 43% of boys and 73% of girls did not meet the PA recommendations of at least 60 minutes/day of MVPA using a accelerometer device [START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF].

Childhood and adolescence are key periods for education about and prevention of adult metabolic diseases. Adolescence is a period marked by a decline in participation in physical activity (PA) mainly because of the reduced time spent in MVPA (Ortega et al., 2013). Many factors are responsible for this decline, including overweight and obesity, social context (e.g., the perceptions and behaviors of siblings, parents or friends), parental educational level, the built environment, and seasonal variation [START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF][START_REF] Valery | Diet, physical activity, and obesity in school-aged indigenous youths in Northern Australia[END_REF][START_REF] Beets | Social support and youth physical activity: the role of provider and type[END_REF][START_REF] Salvy | Peer influence on children's physical activity: an experience sampling study[END_REF][START_REF] Vanhelst | A favorable built environment is associated with better physical fitness in European adolescents[END_REF], Gracia-Marco et al., 2013).

Children and adolescents spend a substantial proportion of their time at school, and intervening in the school environment may provide opportunities for children and adolescents to be more physically active. Recess, after-school programs and commuting to school in walking are opportunities to improve PA levels and other health-related aspects in adolescents [START_REF] Beets | After-school program impact on physical activity and fitness: a meta-analysis[END_REF][START_REF] Saksvig | Travel by walking before and after school and physical activity among adolescent girls[END_REF][START_REF] Saksvig | A cross-sectional and longitudinal study of travel by walking before and after school among eighth-grade girls[END_REF][START_REF] Ridgers | Physical activity during school recess: a systematic review[END_REF]. While several studies have investigated separately the impact of specific periods (recess, after school, commuting to school) on PA patterns during a school day, no study was performed to assess the relationship the school rhythms and the PA patterns and sedentary behaviours in adolescents. The school rhythm corresponds to the organization of the time during school day and includes beginning and finishing hours of the class, number and duration of recess, lunch break, number of school days per week and total time spent at school [START_REF] Dobbins | School-based physical activity programs for promoting physical activity and fitness in children and adolescents aged 6 to 18[END_REF]. It is of public health interest to study whether the school rhythm is associated with PA patterns and sedentary behaviors in adolescents. If so, adaptation of the school rhythm may be a good strategy for promoting PA.

The aim of this study was to assess the relationships between school rhythm and PA patterns, and sedentary behaviors in a large sample of European adolescents.

Methods

Study Design

The current report is based on data from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study. The aim of the HELENA study was to obtain a broad range of standardized, reliable, and comparable nutrition-and healthrelated data from a random sample of European adolescents aged 12.5-17.5 years.

Data were collected in 10 European cities: Vienna (Austria), Ghent (Belgium), Lille (France), Athens (Greece), Heraklion (Greece), Pecs (Hungary), Rome (Italy), Dortmund (Germany), Zaragoza (Spain), and Stockholm (Sweden). In total, 3528 adolescents (1844 girls and 1684 boys) meeting the inclusion criteria completed all examinations. A detailed description of the HELENA study methodology and sampling has been published elsewhere [START_REF] Béghin | Main characteristics and participation rate of European adolescents included in the HELENA study[END_REF][START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF]. In summary, an extended and detailed manual of operations was designed for and thoroughly read by every researcher involved in fieldwork before the data collection started [START_REF] Nagy | Harmonization process and reliability assessment of anthropometric measurements in a multicenter study in adolescents[END_REF]. In addition, a workshop training week was carried before the study began, to standardize and harmonize the methodology of data collection.

The instructions given to the participants in every measurement were standardized for all the cities and were translated into the local language. In this way, the same verbal information was given to all participants in the HELENA Study. Pilot studies were conducted in 10 cities and included 202 adolescents and to assess the intraobserver and interobserver technical error of measurements as well as the reliability of measurements were checked. The detailed information has been previously reported [START_REF] Nagy | Harmonization process and reliability assessment of anthropometric measurements in a multicenter study in adolescents[END_REF].

Written, informed consent was obtained from the adolescent and the parents. The HELENA study was approved by the local ethics committee for each country, and all procedures were performed in accordance with the ethical standards of the Helsinki Declaration of 1975 as revised in 2008 [START_REF] Béghin | Quality assurance of ethical issues and regulatory aspects relating to good clinical practices in the HELENA Cross-Sectional Study[END_REF].

From the total population of 3528 adolescents, 2024 (57.4%) were included in the present study because complete information about their school schedules PA and sedentary behaviors assessment.

Measurements

Participants' Characteristics Anthropometric measurements were carried out only once. Body weight was measured with the participant wearing light clothes, without shoes, to the nearest 0.1 kg using an electronic scale (SECA 871; SECA, Hamburg, Germany). Height was measured without shoes to the nearest 0.1 cm using a telescopic height-measuring instrument (SECA 225; SECA). Body mass index (BMI) was calculated as weight in kg/height in m 2 (kg/m 2 ). Weight status was assessed using the International Obesity Task Force scale [START_REF] Cole | Establishing a standard definition for child overweight and obesity worldwide: international survey[END_REF]. Pubertal status was assessed by a physician through direct observation according to the method of Tanner and Whitehouse [START_REF] Tanner | Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty[END_REF].

Parental education level was classified into one of three categories using a specific questionnaire adapted from the International Standard Classification of Education (ISCED) (http://www.uis.unesco.org/Library/Documents/isced97-en.pdf). Parental education level was scored as I for primary and lower education (levels 0, 1, and 2 in the ISCED classification), II for higher secondary (levels 3 and 4 in the ISCED classification), and III for tertiary (levels 5 and 6 in the ISCED classification).

School Rhythm

Adolescents were recruited through the schools where the entire classes including adolescents aged 13 to 16 years old were asked to participate. Sampling was stratified and random, and was performed in multiple steps [START_REF] Béghin | Main characteristics and participation rate of European adolescents included in the HELENA study[END_REF].

Firstly, within the selected cities, schools were randomized taking into account several cluster stratifications: private/public school, location/area (zone or district), socio-economic level and age strata. The second step included randomisation of classes. In this random cluster sampling process, stratification was done at two levels, first at the level of cities and secondly at the level of school strata (as mentioned above). Data concerning public/private school status, number of adolescents per class, and class level or grade, were provided by local school authorities. This procedure was carried out to ensure diversity of the sample in cultural and socioeconomic aspects. The random selection of schools and classes was performed centrally (by the Ghent University). A list of 10 randomly selected schools was provided for each center. At the same time, a replacement list including 20 substitute schools was also provided (replacement schools/classes were for the event of a school/class refusing to participate, and were from the same district and same class level/grade). Ten schools were involved in the survey for each center. A researcher from each center was instructed to obtain the schedule of the different classes that participated in the study. The questionnaire was completed by the school executive and comprised several parts: (i) beginning and finishing hours of the class every day; (ii) teaching hours (i.e time spent in class only) per day and per week for each class; (iii) hours of recess defined as time in school out of class (including recess per se, commuting in school and lunch break); (iv) number of school days per week; and (v) total amount of time spent at school. All of these parameters were reported for each class during 1 week.

Because there are no validated data for defining school rhythm, we applied an a priori definition of school rhythm using the information contained in the school schedule (Figure 1). Using the information in the diary for each class, we decided to classify a priori the adolescents into two groups: a short school rhythm group and a long school rhythm group. A short school rhythm was defined as finishing school at 3:00 PM or earlier (Figure 1). A long school rhythm was defined as finishing school after 3:00 PM (Figure 1). The two groups differed significantly on other criteria: duration of school recess, total time spent at school per day and per week, hours of teaching per day and per week, and the number of classes with <5 days of school per week (Table 1). School recess was defined as noncurricular time between lessons allocated by schools for the students to engage in leisure activities [START_REF] Ridgers | Physical activity levels of children during school playtime[END_REF]).

PA

The ActiGraph ® Monitor (ActiGraph, GT1M ® model, Pensacola, FL) was used to assess the PA level in free-living conditions. This device has been validated against oxygen consumption and heart rate to assess PA level [START_REF] Vanhelst | New validated thresholds for various intensities of physical activity in adolescents using the Actigraph accelerometer[END_REF][START_REF] Treuth | Defining accelerometer thresholds for activity intensities in adolescent girls[END_REF]. The interinstrument reliability of this device is high for both sedentary and vigorous activities [START_REF] Vanhelst | Comparative interinstrument reliability of uniaxial and triaxial accelerometers in free-living conditions[END_REF][START_REF] Mcclain | Actigraph accelerometer interinstrument reliability during free-living in adults[END_REF]. A researcher of each center involved in fieldwork completed a workshop training about the instructions to give to participants. The adolescents were instructed to wear the accelerometer on the lower back with an elastic belt and adjustable buckle the most longtime possible during 7 consecutive days and they were also asked to follow their normal daily routine. In addition, the participants were instructed to remove the accelerometer during swimming, showering, and bathing, and at night. The epoch interval for the accelerometer was set at 1 min, and the output was expressed as mean counts per minute.

Data were uploaded from the monitor to a computer after the completed 7-day registration period. Participants who did not record at least 3 days (week day and weekend day) of recording with a minimum of 10 h of activity per day were excluded from the analyses (Masse et al., 2005;[START_REF] Ward | Accelerometer use in physical activity: best practices and research recommendations[END_REF]. The assessment of time spent in sedentary activities, moderate PA, vigorous PA, and MVPA was based on the cutoff points of 0-400, 1901-3918 and >3918 counts/min, respectively we previously established in laboratory conditions [START_REF] Vanhelst | New validated thresholds for various intensities of physical activity in adolescents using the Actigraph accelerometer[END_REF]. MVPA was dichotomized into <60 min/day and ≥60 min/day, according to the current PA recommendations [START_REF] Cale | Exercise recommendations for young people: An update[END_REF]. The PA patterns were analyzed according to school days (Monday to Friday) and school-free days when adolescents did not have lessons (Sunday and Saturday and/or Wednesday for some adolescents who did not have lessons). For this study, only cumulative amounts of MVPA were considered.

Sedentary Behaviors

Sedentary behaviors were assessed using a structured questionnaire that included questions about the amount of time habitually spent in front of the television or a computer, or playing video games during school days and school-free days. The questionnaire used questions such as: "On weekdays, how many hours do you usually spend watching television?", "On weekdays, how many hours do you usually spend on computers?", and "On weekdays, how many hours do you usually spend playing video games?" The answers were classified into two categories: 0-2 h/day and >2 h/day [START_REF] Strong | Evidence based physical activity for school-age youth[END_REF][START_REF] Dunstan | Television viewing time and mortality: the Australian Diabetes, Obesity and Lifestyle Study (AusDiab)[END_REF]. The questionnaire was administered in the classroom under the supervision of an HELENA fieldworker during the morning before the PA assessment. The complete duration of this test was 10 minutes. This measure has been shown to provide a reliable (intraclass correlation=0.82; 95% CI 0.75 to 0.87) and valid (criterion validity=0.3) [START_REF] Strong | Evidence based physical activity for school-age youth[END_REF][START_REF] Dunstan | Television viewing time and mortality: the Australian Diabetes, Obesity and Lifestyle Study (AusDiab)[END_REF]. This questionnaire was used for studying the impact of school rhythm on sedentary behaviors.

Statistical Analysis

Data are presented as number (percentage) for qualitative variables and as mean (± standard deviation [SD]) or median (range) for quantitative variables. The normality of the distribution was checked graphically and using the Shapiro-Wilk test. Bivariate comparisons between the two school rhythm groups were made using the Student t test or Mann-Whitney U test for quantitative variables, chi-square test for nonordinal qualitative variables, and a Mantel-Haenszel trend test for ordinal qualitative variables. Comparisons for PA and sedentary outcomes between the two school rhythm groups were adjusted for prespecified confounding factors such as age, sex, BMI, pubertal status, and mother's and father's educational levels [START_REF] Ridgers | Physical activity during school recess: a systematic review[END_REF][START_REF] De Moraes | Independent and combined effects of physical activity and sedentary behavior on blood pressure in adolescents: gender differences in two cross-sectional studies[END_REF]Rey-Lopéz et al., 2011, Ortega, Ruiz, & Castillo, 2013). Adjustment of the prespecified confounding factors was made using analysis of covariance for quantitative outcomes (for total PA, MVPA, and sedentary PA) and logistic regression analysis for binary outcomes (for ≥60 min/day of MVPA during the week and sedentary behaviors). Odds ratios (ORs) with their 95% confidence intervals (95% CIs) for each binary outcome were calculated using the long rhythm group as the reference. All statistical tests were performed using a two-tailed α level of .05. The data were analyzed using SAS software version 9.4 (SAS Institute Inc., Cary, NC).

Results

This study included 2024 adolescents (1008 boys and 1015 girls). The mean age, height and body weight were 14.6 ± 1.2 years, 165.7 ± 9.2 cm and 58.4 ± 12.4 kg, respectively. The median [range] time spent at school, in class and in recess were 6.10 [4.00; 9.55] hours per day, 5.40 [3.00; 8.15] hours per day and 60 [15; 150] min per day, respectively.

The short rhythm group comprised those students who finished school earlier in the afternoon (before 3:00 PM) and who had a shorter cumulative school recess and a shorter time spent at school during the week. The long rhythm group comprised those students who finished later in the afternoon (after 3:00 PM) and who had a longer time spent in the school environment and a longer cumulative school recess (Table 2). In average, students in the short rhythm group finished 2.20 hours earlier, had 50 min per day less time in recess and spent 9 hours per week less time in the school environment compared with those in the long rhythm group.

Among the 2024 adolescents with available data about their school schedules, 794 (39.2%) had a long school rhythm. The long rhythm group had a higher percentage of girls, older age, lower adiposity levels, and parental education level compared with the short rhythm group.

PA

The distribution of objective PA measurements throughout the week varied significantly between the two rhythm groups (Table 3) before and after adjustment for the prespecified confounding factors.

The short rhythm group had a lower number of counts during the week compared with the long rhythm group. Analysis of the number of counts according to the different daytime activities showed that this difference reflected differences in the number of teaching and recess hours: the short rhythm group had a higher number of counts during hours spent outside school (school-free days or during school days) compared with the long rhythm group.

The amount of MVPA also differed between the two groups, although the difference was not significant on school-free days (Table 3). After adjusting for prespecified confounding factors, students in the short rhythm group spent 9.1% less time in MVPA per week compared with those in the long rhythm group (P < .0001).

The corresponding differences for MVPA during teaching and recess hours were 25.2% and 62.3%, respectively.

Students in the short rhythm group were less likely to meet the PA recommendation of ≥60 min/day of MVPA per week compared with students in the long rhythm group (30.7% vs. 34.1%; adjusted OR, 0.73; 95% CI, 0.58-0.92; P = .008).

Students in the short rhythm group spent more time in sedentary activities per week than did those in the long rhythm group (mean ± SD, 860 ± 52 vs. 848 ± 49 min/day; P < .0001). This difference was explained mainly by the hours spent outside school (565 ± 53 vs. 464 ± 53 min/day; P < .0001), whereas no such difference was found for other daytime activities. The short rhythm group spent significantly less sedentary time during teaching and recess compared with the long rhythm group. When expressing MVPA in percentage according to teaching and recess duration, we found that the percentage of MVPA of school attendance was higher in the long rhythm group both during recess and teaching hours compared to the short rhythm group. Similarly, the percentage of time spent in sedentary PA during the school attendance was higher in the long rhythm group compared to the short rhythm group (Table 3).

Sedentary Behaviors

Sedentary behaviors during school days and school-free days differed between the two rhythm groups (Figure 2). During school days, a higher percentage of students in the short rhythm group watched television or played video games for >2 h/day compared with the long rhythm group. The time spent on a computer did not differ significantly between groups. After adjusting for the prespecified confounding factors, the ORs in the short rhythm group were 1.54 (95% CI, 1.22-1.94) for spending ≥2 h/day watching television and 2.08 (95% CI, 1.22-1.94) for spending ≥2 h/day playing video games.

During school-free days, a higher percentage of students in the short rhythm group also watched TV or played video games for >2 h/day compared with the long rhythm group. However, after adjusting for the prespecified confounding factors, only the difference in the percentage of students who played video games for >2 h/day remained significant (OR, 1.60; 95% CI, 1.15-2.24). By contrast, a lower percentage of students in the short rhythm group spent >2 h/day on a computer (65.3% vs. 73.1%; adjusted OR, 0.71; 95% CI, 0.56-0.89).

Discussion

School rhythms correspond to alternating lessons and noncurricular periods including recess, break lunch and extracurricular activities. Although each country defined its own directives for organizing school time, many school rhythms vary among different schools of the same country as shown in our study (Figure 1). To our knowledge, this is the first study to assess whether the school rhythm is associated with PA patterns and sedentary behaviors in European adolescents from 7 countries. The main finding was that a long school rhythm was associated with higher PA levels, mainly during school recess, and less time spent in sedentary activities. Another important result is that adolescents who had a long school rhythm also had a higher PA level over the entire week and were more likely to meet the PA recommendations. These results show the importance of both the school recess and after-school time in promoting PA to adolescents.

In our study, students in the schools with the shorter rhythm spent half the time in recess during a school day compared with those in the schools with a long rhythm. In addition, we showed that the time spent in MVPA during recess was lower in the short rhythm group than the long rhythm group reinforcing the effect of recess duration on PA. In a recent systematic review, Ridgers et al. showed that recess in schools contributes strongly to fulfilling the daily recommended amount of MVPA for adolescents [START_REF] Ridgers | Physical activity levels of children during school playtime[END_REF]. Despite the identified decline in PA and the increase in sedentary time during recess, recess time contributes to about 20% of the recommended daily MVPA level when no interventions are used [START_REF] Martinez-Gomez | Physical Activity During High School Recess in Spanish Adolescents: The AFINOS Study[END_REF]. Intervention studies of sedentary and active behaviors have shown positive effects of an intervention program during recess [START_REF] Kriemler | Effect of school-based interventions on physical activity and fitness in children and adolescents: a review of reviews and systematic update[END_REF][START_REF] Toftager | Intervention effects on adolescent physical activity in the multicomponent SPACE study: a cluster randomized controlled trial[END_REF]. Our results indicate that providing recess time and encouraging PA in schools with a shorter school rhythm may increase MVPA. In addition to physical education classes, the school recess is one of the main opportunities for children and adolescents to be physically active in the school environment. As more MVPA was performed during teaching hours, we cannot excluded that long rhythm group had more physical education lessons compared to short rhythm group. This hypothesis could not be confirmed since we had no precise information regarding sport participation (out and at school) in our population.

Another outcome from our study is that adolescents in the short rhythm group who finished school earlier in the day were more likely to perform sedentary activities (watching TV, playing video games or computer). A possible explanation is that the highly condensed teaching hours (with short breaks between lessons) leads to tiredness at the end of the school day, which increases sedentary behaviors after school hours. This hypothesis remains to be tested in an appropriate intervention study. Previous studies showed that after-school programs contribute to increase the time spent in MVPA during school days [START_REF] Lubans | Evaluation of an extra-curricular school sport programme promoting lifestyle and lifetime activity for adolescents[END_REF][START_REF] Weintraub | Team sports for overweight children: the Stanford Sports to Prevent Obesity Randomized Trial (SPORT)[END_REF][START_REF] Kelder | The CATCH Kids Club: a pilot after-school study for improving elementary students' nutrition and physical activity[END_REF]. [START_REF] Lubans | Evaluation of an extra-curricular school sport programme promoting lifestyle and lifetime activity for adolescents[END_REF] demonstrated that extracurricular school sport program permits to increase of 34% the PA level at the end of program. In a meta-analysis, Beets et al. showed also that interventional after-school programs have a positive impact on different health outcomes (reduce sedentary behaviors and BMI, increase MVPA and physical fitness) [START_REF] Beets | After-school program impact on physical activity and fitness: a meta-analysis[END_REF]. Our results are consistent with previous studies and suggest that the after-school setting holds considerable promise for increasing the PA levels of youth. [START_REF] Lubans | Evaluation of an extra-curricular school sport programme promoting lifestyle and lifetime activity for adolescents[END_REF][START_REF] Weintraub | Team sports for overweight children: the Stanford Sports to Prevent Obesity Randomized Trial (SPORT)[END_REF][START_REF] Kelder | The CATCH Kids Club: a pilot after-school study for improving elementary students' nutrition and physical activity[END_REF].

Our results also show that the school rhythm influences PA both on school days and over the entire week. Moreover, our finding shows that PA (both MVPA and sedentary) did not differ during school free days between the two groups, suggesting that the number of day in class (< or ≥ 5 days of school per week) did not influence the results we observed. This is inconsistent with the hypothesis of an "activity stat," which says that increasing PA during school hours will not increase the overall PA level because of the actions of a biological PA regulator through which the body uses a range of biological responses to changes in PA to maintain a constant daily energy expenditure [START_REF] Metcalf | Physical activity cost of the school run: impact on schoolchildren of being driven to school (EarlyBird 22)[END_REF][START_REF] Wilkin | Can we modulate physical activity in children? No[END_REF]. Several studies have found no evidence for same-day or next-days in the amount of PA performed [START_REF] Long | School-day and overall physical activity among youth[END_REF]Goodman, Mckett, & Paskins, 2011[START_REF] Baggett | Compensation or displacement of physical activity in middleschool girls: the Trial of Activity for Adolescent Girls[END_REF].

In our study, the difference in MVPA we found between the 2 groups 7% that rise the question of clinical significance of such quite low difference. There no clear answer to this important question [START_REF] Wen | Minimum amount of physical activity for reduced mortality and extended life expectancy: a prospective cohort study[END_REF][START_REF] Hupin | Even a low-dose of moderate-to-vigorous physical activity reduces mortality by 22% in adults aged ≥60 years: a systematic review and meta-analysis[END_REF] although recent studies clearly show a progressive continuous positive effect of MVPA on mortality rate, suggesting that even small difference at a population could have a positive effect on global health indicators.

Our study has strengths and limitations. One strength is that it included a large sample size of adolescents and used a panel of standardized and harmonized tests to assess nutrition and PA level (i.e., accelerometry is a robust objective method for evaluating PA patterns) [START_REF] Moreno | Design and implementation of the Healthy Lifestyle in Europe by Nutrition in Adolescence Cross-Sectional Study[END_REF]. One limitation is the subjectivity of the assessment of the sedentary behaviors, which were evaluated and reported by the adolescents themselves. Another limitation is the cross-sectional design of the study, which rules out the ability to draw causal conclusions. In that, setting the two groups differed by several characteristics. However, we were able to control for several confounding factors (age, sex, BMI, pubertal status, and mother's and father's educational levels) in the statistical analysis. Nevertheless, since the number of physical education lessons and after-school activities were not available, we could not introduced them in the statistical analysis although they could have an influence on our results. Regarding the multiple comparisons issues, we could not also exclude false significant association and the present results should be interpreted with caution. The study is also limited by the use of only a single week of measurement of PA and school schedules. However, in European countries, the school schedule is the same throughout the school year. Variations in weather (wind, rain and sunshine) during the PA assessment were not recorded in our study and might also have affected our results. Finally, since short and long rhythm group differs also by center/country, we cannot exclude the effect of different schools culture of spending the recess.

Conclusions

Our results suggest that school time provides a good opportunity for adolescents to be physically active. Sedentary behavior was observed mainly during school-free periods, which suggests that after-school programs may be complementary and beneficial for promoting PA. The data obtained from this study contribute to a better understanding of the PA patterns in adolescents and may help caregivers and health and school authorities adapt school rhythms for the promotion of PA. Odds-ratio (OR) of each sedentary outcomes was calculated for short rhythm group using long rhythm group as reference. * Adjusted for sex, age, BMI, pubertal status and parental educational levels.

Legends

Figure 1 .

 1 Figure 1. Description of school diaries for each class studied

Figure 2 .

 2 Figure 2. Impact of school rhythm on sedentary behavior throughout the week

Figure 2 .

 2 Figure 2. Impact of school rhythm on sedentary behavior throughout the week

Table 1 .

 1 Characteristics of school diaries for the two school rhythm groups

Table 2 .

 2 Adolescent's characteristic according to school rhythm group

Table 3 .

 3 Impact of school rhythm on physical activity levels throughout the week

Funding

The HELENA study is made possible by the financial support of the European Community Sixth RTD Framework Programme (Contract FOOD- CT-20056007034) and the Spanish Ministry of Science and Innovation (RYC-2010-05957 and RYC-2011-09011). The content of this paper reflects only the authors' views, and the European Community is not liable for any use that may be made of the information contained therein.

Conflict of interest

The authors do not have any competing interests.


Claude Saint-Ruf

Oskar Lechner

Jacqueline Feinberg

Harald Von Boehmer

Genomic structure of the human pre-T cell receptor > chain and expression of two mRNA isoforms

Keywords: pT §, Genomic structure, mRNA isoform, T cell development, Gene expression

The pre-TCR, which is minimally composed of the TCR g chain, the pre-T § chain, and the CD3 complex, regulates early T cell development. The pre-T § chain is a 33-kDa type I transmembrane glycoprotein with an extracellular part similar to the constant domain of the immunoglobulin supergene family. We have sequenced (11 kb) the human pT § gene, which like the murine pT § gene consists of four exons: exon 1 encodes the 5' untranslated region, the leader peptide and the first three amino acids of the mature protein, exon 2 the extracellular immunoglobulin (Ig)-like domain, exon 3 a 15-amino acid peptide including a cysteine required for heterodimerization with TCR g , exon 4 the transmembrane region, the cytoplasmic tail and the 3' untranslated sequence. The human pT § gene is located on chromosome 6p21.3, close to the HLA-A locus. Reverse transcription-PCR studies with human thymus and leukemic cells showed that alternative splicing produces a shorter pT § isoform, which lacks the Ig-like domain but contains the transmembrane elements and the extracytoplasmic cystein and which could thus permit pairing with TCR g chain and association with CD3 molecules. The conserved splice sites suggest a yet ill-defined biological function of the short pT § protein.

Introduction

In the thymus T cells develop from precursor cells in discrete steps that are controlled by the pre-TCR as well as the § g TCR for antigen. The pre-TCR consists minimally of the TCR g chain and the covalently bound pre-TCR § chain associated with signal-transducing proteins of the CD3 complex [START_REF] Groettrup | A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein[END_REF]. The pre-TCR rescues developing T cells that harbor a productive TCR g rearrangement from programmed cell death and induces extensive cell division as well as differentiation. It is also essential for allelic exclusion of the TCR g locus through feedback inhibition of TCR g rearrangement (reviewed in [START_REF] Von Boehmer | Structure and function of the pre-T cell receptor[END_REF]). Finally, it appears to play an important role in directing T cell development into the § g pathway [START_REF] Aifantis | Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor beta locus[END_REF].

Both murine [START_REF] Saint-Ruf | Analysis and expression of a cloned pre-T-cell receptor gene[END_REF] and human [START_REF] Del Porto | Cloning and comparative analysis of the human pre-T-cell receptor alpha chain gene[END_REF] pT § cDNA have been cloned. The deduced amino acid (aa) sequences revealed a type I transmembrane protein consisting of a hydrophobic signal peptide, an extracellular domain with a structure similar to the constant (C) domain of the immunoglobulin (Ig) supergene family, hydrophobic transmembrane region, and a cytoplasmic tail. In contrast to the short (3 aa) cytoplasmic domains of the TCR § and TCR g chain, pT § has a prominent cytoplasmic tail (30 aa in mice and 114 aa in humans). Additionally, the pT § is not subjected to the rearrangement process and it is devoid of J-like sequences as well as V-like Ig domains [START_REF] Saint-Ruf | Analysis and expression of a cloned pre-T-cell receptor gene[END_REF][START_REF] Fehling | Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene[END_REF]. With the exception of the cytoplasmic domain, murine and human pT § cDNA are very homologous (70-80 %). Murine pT § is located in the D/E1 region on chromosome 17. The human gene resides in the syntenic region p21.2-p12 on chromosome 6 close to the MHC.

In the present study we present data on the human pT § gene cloned from a human chromosome 6 cosmid library. To define some conserved regions potentially important for gene regulation the complete human pT § gene sequence was established and compared with the murine sequence. Analysis of pT § expression in human thymus and in leukemic human cell lines shows that a splice variant pT § isoform is expressed. This isoform, which lacks the Ig-like domain could also be found in mice and therefore may have an important function in T cell development. A cosmid library of human chromosome 6 DNA was purchased from the Reference Library Database (RFLB) of the Max Planck Institute for Molecular Genetics (Berlin, Germany). Nizetic et al. [START_REF] Nizetic | Construction of cosmid libraries from flow-sorted human chromosomes 1, 6, 7, 11, 13, and 18 for reference library resources[END_REF] constructed the library from DNA digests of flow-sorted human chromosomes 6 of the 4× cell line which were ligated into lawris 4. DH5 alpha MCR (BRL) was used as host. Subsequently, a high-density colony filter ( ˚36 864 clones) corresponding to this library, was hybridized with a human pT § cDNA probe. Ten clones were found to be pT § positive. One of them co-hybridizes with a probe designated Alu yac 7 (RFLD data), which is ALU PCR-derived genomic. This clone is HLA-A positive and located on 6p21-3 [START_REF] Shortle | A cosmid library specific for human chromosome regions 6p21.3 and 6q27[END_REF], thereby indicating the localization of the pT § gene. Previous in situ hybridization data already mapped it to the 6p21-6p12 region of the human genome [START_REF] Del Porto | Cloning and comparative analysis of the human pre-T-cell receptor alpha chain gene[END_REF].

Positive clones for pT § were mapped by restriction digestion and Southern blotting. Three of them, containing the complete pT § gene, were extensively studied. No difference was found regarding the organization of pT § in these three recombinants. A pT § restriction map is shown in Fig. 1. For two directional sequencing we subcloned inserts of 34 kb, 44 kb, and 46 kb, respectively into bluescript KS + plasmids.

Sequence and organization of the human pT > gene

Different subclones were sequenced using the "abiprism" system (PE Applied Biosystems, GB). We sequenced 11 kb starting 1500 bp upstream the ATG codon and ending 250 bp downstream the stop codon (se-quence is available under accession number AF084941).

Comparing this genomic sequence with pT § cDNA revealed the following exon/intron organization (Fig. 2).

Human pT § is encoded by four exons, which correspond, as in most other Ig superfamily genes, approximately to the different domains of the protein. The same organization has been observed for murine pT § . The first exon contains the 5' untranslated sequence, the leader peptide and the first three aa of the mature protein. In humans the following intron 1 is 6.5 kb long (5 kb in mice). It is particularly rich in repetitive DNA: 16 SINE elements (16 ALU sequences) and 3 LINE elements (2 LINE-2 and 1 LINE-1). Five simple repeats and 2 low complexity sequences have also been noticed. Exon 2 encodes 105 aa of the extracellular Ig-like domain, including the two cysteines that are thought to form the typical Ig intrachain disulfide bridge. It is followed by intron 2 (887 bp) containing one SINE element (mir) Exon 3 encodes 16 aa, which form the connecting peptide con-taining the cysteine residues for dimerization with the TCR g chain. Exon 2 and exon 3 have the same size in humans and mice. The following intron contains 1057 bp encompassing a SINE element (Alu Sg). Exon 4 encodes 139 aa, and contains the sequence for the approximately 20-aa long transmembrane region and the 3' untranslated region of ˚160 nucleotides. This includes two positively charged aa (arginine, lysine) which are located at the same position in the murine pT § chain but also in the TCR § and TCR ˇchain. These arginine and lysine are thought to be essential for the interaction of the TCR chains with negatively charged residues in the transmembrane regions of the CD3 complex [START_REF] Cosson | Membrane protein association by potential intramembrane charge pairs[END_REF]. The last 114 aa form the cytoplasmic tail. While more than 80 % aa identity can be observed between the murine and human Ig-like and transmembrane region, no significant homology could be found between the 114-aa human cytoplasmic tail and the 30 residues of the murine pT § cytoplasmic tail. We have suggested [START_REF] Del Porto | Cloning and comparative analysis of the human pre-T-cell receptor alpha chain gene[END_REF][START_REF] Fehling | Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene[END_REF] that the lack of identity between the human and murine cytoplasmic tails could reflect a divergence in gene structure. In fact, the organization of the pT § genome is very similar to the constant regions of TCR § and TCR ˇgenes. However, two differences can be found. First, only pT § is endowed with a leader sequence located far from the next coding sequence. This could be explained by the fact that pT § is expressed without a second, V region-like Ig domain.

Second, the pT § transmembrane region, the cytoplasmic tail, and the 3' untranslated region are encoded by a single exon. In TCR § and TCR ˇgenes the 3' untranslated region is encoded by a separate exon. In mice the 3' untranslated region contains a B2 repetitive element which provides the polyadenylation site and thus can be considered as an integral part of the pT § gene. We have suggested that the specific structure of the murine pT § gene could be due to the presence of this transposonlike element in the 3' end of the gene [START_REF] Fehling | Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene[END_REF]. Because human pT § has the same organization but has lost the B2 repetitive elements, this assumption is no longer valid.

Regulatory elements of the pT > gene

We sequenced (1.5 kb) upstream of the ATG start codon of the pT § gene. Usually promoter sequences are present within 100 bp upstream of the ATG start codon. While this region is well conserved between humans and mice (70 % of nucleotide identity) no TATA or CCAAT like regulatory elements, which are typical promoter motifs of many non-housekeeping genes, could be found. Furthermore, computer-based sequence analysis did not permit to define a potential promoter. Further analyses of this region by deletion and/or mutagenesis experiments will be necessary to define the promoter site and further regulatory elements.

Intron 1 is very large compared to other Ig-family genes (6.5 kb for the human and 5 kb for the murine gene). In the middle of this intron a single, 80-bp long sequence is highly conserved between humans and mice (80 % homology). Since intronic sequences are usually not conserved between species, except if they contain some regulatory elements, this sequence could play a role in the regulation of pT § . A detailed analysis using the TESS program (BCM Search Launcher) revealed potential binding sites for transcription factors AP1 and ? -EBP-C2. AP1 family members are known to play a critical role in regulating T cell gene expression in combination with other transcription factors [START_REF] Leiden | Transcriptional regulation of T-cell genes during T-cell development[END_REF]. Binding sites for ? -EBP-C2 have been found in intragenic enhancers of TCR g and IgH [START_REF] Peterson | Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms[END_REF][START_REF] Mcdougall | A transcriptional enhancer 3' of C beta 2 in the T cell receptor beta locus[END_REF]. Like in mice, the human pT § intron 1 is heavily loaded with short interspersed repetitive elements. About 4.5 kb of the 6.6 kb of human intron 1 are repeat sequences. The conservation of these sequences among humans and mice additionally suggests a role of intron 1 in the regulation of pT § expression. Since LINE elements carry an internal split promoter for RNA polymerase III, they are actively transcribed by this polymerase. They participate in host gene activity regulation by inserting into regulatory elements or by providing regulatory elements themselves [START_REF] Georgiev | Mobile genetic elements in animal cells and their biological significance[END_REF][START_REF] Berger | Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 ˇis physically associated but not functionally required[END_REF]. Therefore, it would be interesting to analyze whether this region plays an important role in the regulation of pT § expression. This could be helpful to understand the commitment of T cells to either the § g or the + ˇlineage since it appears that + precursors but not + ˇT cells express the pT § gene [START_REF] Aifantis | Essential role of the pre-T cell receptor in allelic exclusion of the T cell receptor beta locus[END_REF].

Expression of human pT > and evidence for a splice-variant

We have used RT-PCR to analyze expression of human pT § in human thymus and in the human HPB-ALL, MOLT-4 and Jurkat cell line. Primers have been chosen in exon 1 and exon 4 in order to amplify the entire cDNA. Specific transcripts were found in the human thymus, in HPB-ALL and MOLT-4 cells, respectively. The Jurkat cell line was pT § negative. In all positive samples we observed two different bands (Fig. 3). One of the expected size of ˚640 bp, and a smaller one of ˚320 bp. Sequencing of these bands revealed that the larger one corresponds to the originally described form of pT § (as described in [START_REF] Del Porto | Cloning and comparative analysis of the human pre-T-cell receptor alpha chain gene[END_REF]), designated pT § 1. The smaller band represents a splice-variant of pT § , designated pT § 2.

The splice variant sequence, pT § 2, shows an open reading frame. The deduced primary sequence contains 231 aa. Comparison with pT § 1 revealed that pT § 2 lacks the major part of the Ig-like domain. During pT § 2 splicing, which uses at the same splice sites as pT § 1, the [START_REF] Hounsell | Olinked glycosylation structure and function[END_REF]). As no consensus sequence(s) for O-glycosylation sites could be defined up to now, further analyses are needed to establish possible glycosylations.

Immunoprecipitation of the human pT > protein

The human cell lines HPB-ALL and Jurkat, which express and do not express the two pT § isoforms, respectively, were labeled with [ 3 H]leucine. Lysates were immunoprecipitated with R225 antibodies. These polyclonal rabbit antibodies were generated against the complete extracellular domain of murine pT § including the connecting peptide. The antibodies recognize the protein generated in vitro from human pT § 1 cDNA by an eukaryote-coupled transcription/translation system sup-plemented with dog pancreas microsomes (data not shown). Precipitates were analyzed by SDS-PAGE. A 33-34-kDa protein could be detected in precipitates from HPB-ALL but not from Jurkat cells (Fig. 4). This protein is likely to correspond to pT § 1. The same results were obtained under reducing and nonreducing conditions and suggest that in HPB-ALL cells pT § 1 is not linked to another protein. The human pT § 2 isoform could not be detected in these experiments. It is possible, however, that the R225 antibodies do not recognize this form. In contrast to the Ig-like domain, which shows 85 % homology between mice and humans and is only present in pT § 1, only 55 % homology exists between the murine and human 20 aa extracellular part which is shared by the two pT § isoforms. Thus, the R225 antibodies may not bind to the connecting peptide (compared to the Ig-like domain) of human pT § .

We were unable to precipitate pT § in biotinylated surface HPB-ALL cell extracts. In this cell line, which expresses the TCR § g , pT § should not be expressed within a pre-TCR complex on the cell surface.

An additional pT > heterodimer in mice

Previous RT-PCR studies on pT § expression revealed two pT § forms in the murine thymus [START_REF] Bruno | Intra-and extrathymic expression of pre-T cell receptor § gene[END_REF]. Since a corresponding smaller size mRNA could not be detected by Northern blotting we concluded that it was a PCR artifact. However, recently Barder et al. [START_REF] Barber | Cutting edge: The expression in vivo of a second isoform of pT § : implications for the mechanism of pT § action[END_REF] reported the expression of mRNA corresponding to the two pT § isoforms. Prior sequence analysis had revealed that the murine splicing variant had exactly the same organization as human pT § [START_REF] Bruno | Intra-and extrathymic expression of pre-T cell receptor § gene[END_REF].

To analyze whether pT § 2 protein could be detected cell surface biotinylation was performed with the SCB29 cell line [START_REF] Groettrup | T cell receptor (TCR) g chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines[END_REF]. After cell lysis, pre-TCR complexes were immunoprecipitated with an anti-TCR g antibody (F23.1) and analyzed by two-dimensional, reducing and nonreducing, diagonal gel electrophoresis. Gels were blotted and membranes labeled with streptavidin-horseradish peroxidase (HRP) to detect all precipitated proteins. Subsequently, the membrane was reprobed with a pT §specific polyclonal antiserum (R225). Using the anti-TCR g antibody two complexes could be precipitated (Fig. 5A): the first, representing the major complex, consisted of the TCR g chain and the originally described, full-length pT § (pT § 1). The second, weakly detectable complex consisted of a protein with the same size as TCR g , linked to a second protein of 20 kDa (Fig. 5B). This latter protein was recognized by the polyclonal pT § antiserum R225. Since without glycosylation pT § could have an apparent molecular weight of 9 kDa we cannot be certain that a pT § 2 protein is indeed associated with the TCR g chain as has been claimed by Barder et al. [START_REF] Barber | Cutting edge: The expression in vivo of a second isoform of pT § : implications for the mechanism of pT § action[END_REF].

The second heterodimer could also represent a degradation product of the pT § /TCR g heterodimer.

Concluding remarks

Recently, Irving et al. [START_REF] Irving | Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains[END_REF] have shown that thymocytes expressing a pre-TCR lacking the extracellular Ig-like domains of both pT § as well as TCR g develop normally.

They generated RAG -/-transgenic mice expressing a truncated form of the TCR g and the pT § chain, which had their (normal) extracellular domain replaced by a "flag" or "myc" epitope, but still had the cysteine residue necessary for heterodimerization. Thymocyte development could be restored in these transgenic mice. The truncated pT § construct used to generate these transgenic mice is very similar to the variant pT § isoform found in mice and humans but these mice obviously contained also endogenous pT § . Nevertheless, these results were interpreted to indicate that pre-TCR complex without extracellular Ig-like domains could be sufficient to drive the transition from the double-negative to the double-positive stage without binding to an extracellular ligand. Irving et al. [START_REF] Irving | Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains[END_REF] observed that in transgenic RAG -/-mice, that expressed only the truncated TCR g Myc transgene, the modified pre-TCR (composed by the truncated g chain and the endogenous pre TCR § chain) was inefficiently expressed on the cell surface. Based on these results they proposed that only a symmetrical pre-TCR complex can be stable or properly assembled. Following this idea, a pre-TCR complex with pT § 2 should not be stable. For the moment we can only speculate about the possible role of this differential splicing form. It is possible that pT § 2 could compete with pT § 1 for CD3 and TCR g binding, forming an inactive or differentially signaling complex, thus providing a regulatory element in pre-TCR assembly.

Materials and methods

Cell lines and tissues

Three human acute lymphoblastic leukemia cell lines, kindly provided by Dr. MacIntyre (H ôpital Necker, Paris, France), were used for our experiments: the mature, TCR + and CD3 + T cell lines HBL-all and Jurkat, and the + ˇT cell line MOLT4 [START_REF] Minowada | Rosetteforming human lymphoid cell lines. I. Establishment and evidence for origin of thymus-derived lymphocytes[END_REF][START_REF] Fitzgerald | Rhom-2 expression not always correlates with abnormalities on chromosome 11 at band p13 in T cell acute lymphoblastic leukemia[END_REF]. These cell lines were cultured in RPMI 1640/10 % FCS (Gibco-BRL, Gaithersberg, MD). The immature, pre-TCR-positive T cell line SCB29 [START_REF] Groettrup | A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein[END_REF] was cultured in IMDM/10 % FCS (Gibco). A thymus from a 3-month-old child was purchased from H ôpital Saint-Louis (Paris, France).

Isolation, mapping and sequencing of genomic DNA

High-density colony filters corresponding to a cosmid library of human chromosome 6 (no. 109 L4/FS6; Reference Library Database, Max Planck Institute for Molecular Genetics, Berlin; [START_REF] Nizetic | Construction of cosmid libraries from flow-sorted human chromosomes 1, 6, 7, 11, 13, and 18 for reference library resources[END_REF]) were prehybridized overnight at 42 °C in 50 % formamide, 4 × SSC, 50 mM sodium phosphate (pH 6.8-7.2), 1 mM EDTA (pH 8.0), 10 % dextran sulfate, 1 % SDS, 50 ? g/ml denatured salmon sperm DNA and 10× Denhart's solution [START_REF]Molecular cloning: A laboratory manual[END_REF]. Hybridization was performed in the same solution and under the same conditions with an [ § -32 P]dCTP random priming labeled (High-Prime, Boehringer Mannheim), human pT § cDNA probe [START_REF] Del Porto | Cloning and comparative analysis of the human pre-T-cell receptor alpha chain gene[END_REF]. For detection of positive clones, filters were washed three times in 0.1 % SSC at 65 °C and exposed to an X-ray film (Kodak). The pT § cosmids-containing bacterial clones DHR-5 § -MCR were supplied by the Max Planck Institute for Molecular Genetics. Cosmid DNA was purified according to Birnboim and Doly [START_REF] Birnboim | A rapid alkaline extraction procedure for screening recombinant plasmid DNA[END_REF].

DNA inserts were mapped by Southern blotting: endonuclease-digested cosmid DNA samples were subjected to electrophoresis and transferred with 0.

RT-PCR

Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription was carried out using Superscript II reverse transcriptase kit (Gibco). Subsequent PCR was carried out in a total volume of 50 ? l, containing 1 mM deoxynucleoside triphosphates (dNTP), 0.5 ? M of each primer, 5 ? l 10× buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl 2 and 0.01 % (w/v) gelatin, in a volume of 1.5 ml), 2 ? l cDNA from the RT step, and 2 U AmpliTaq® DNA polymerase, LD (PE Applied Biosystems).

After a 3-min denaturation step at 94 °C, 40 cycles consisting of 45 s at 94 °C, 1 min at a primer-specific annealing temperature (T A ), and 1 min at 72 °C were performed in a Gene Amp PCR System 9700 thermal cycler (PE Applied Biosystems). Amplified DNA fragments were analyzed on a 2 % agarose gel in 1× TAE buffer. The PCR primers used for human pT § (T A j 60 °C) were CTGCAGCTGGGTCCTGCCTC and AGTCTCCGTGG-CCGGGTGCA. Primers for human g -actin (T A j 55 °C) were ACACTGTGCCCATCTACGAGGG and ATCATG-GAGTTGAAGGTAGTTTCG.

Protein analysis

L-[4,5-

3 H]-leucine metabolic and cell surface biotinylation were performed as described previously [START_REF] Groettrup | A role for a pre-Tcell receptor in T-cell development[END_REF]. Subsequently, cells were lysed for 1 h in Triton lysis buffer (20 mM Tris/HCl, 150 mM NaCl, 2 % Triton X-100, 1 mM MgCl 2 , pH 8) or digitonin lysis buffer (10 mM triethanolamine, 150 mM NaCl, 1 % digitonin, 1 mM EDTA) supplemented with 5 mg/ml aprotinin, 5 mg leupeptin, 100 mM PMSF and 10 mM iodoacetamide. Nuclei were removed by a 10 min centrifugation at 12 000 × g at 4 °C. The remaining supernatant was precleared four times by a 2-6-h incubation with normal rabbit or mouse serum and protein G-Sepharose CL-4B (pG; Pharmacia, Uppsala, Sweden) in Triton or digitonin lysis buffer.

Proteins were immunoprecipitated by a 3-h incubation at 4 °C with pG-coupled R225 polyclonal antiserum (rabbit anti-murine pT § extracellular domain; [START_REF] Ungewiss | Cloning and expression of a pre-T cell receptor gene[END_REF]) or F23.1 monoclonal antibody (mouse anti-murine TCR g -V g 8). Unbound proteins were removed by washing four times with NET-TON buffer (650 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 0.5 % Triton X-100, 0.05 % NaN 3 , 1 mg/ml ovalbumin) and twice with NET-T (NET-TON with 0.15 M NaCl and without ovalbumin). Proteins were eluted from beads with reducing or nonreducing sample buffer (10 % glycerol, 2.3 % SDS, 62.5 mM Tris-HCl, pH 6.8, 0.05 % w/v bromophenol blue ± 10 mM mercaptoethanol) by boiling for 5 min and subsequent addition of iodoacetamide (60 mM final concentration).

Proteins were run in a one-dimensional 12 % polyacrylamide gel or in two-dimensional diagonal SDSpolyacrylamide gels (nonreducing 8 % polyacrylamide gel in the first and reducing 12 % polyacrylamide gel in the second dimension) as described previously [START_REF] Groettrup | A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein[END_REF]. For visualization of radioactive proteins gels were fixed, incubated in Amplify solution (Amersham), dried and analyzed by autoradiography.

After electrophoresis, samples were transferred onto polyvinylidene difluoride membranes by semi-dry blotting. Membranes were blocked in the following solution: PBS, 4% Tween 20, 2% fetal calf serum 10% non-fatty milk powder. Blots were incubated with streptavidin-HRP conjugate (Southern Biotechnology, Birmingham, AL) for 30 min, at room temperature in PBS 4% Tween 20. HRP was detected by incubation with ECL detection solution (Amersham) and exposure to an X-ray film (Kodak). Blots were destained by incubation in PBS/0.1% sodium azide for reprobing. Subsequently, blots were incubated in PBS 4% Tween 20 with the pT § -specific R225 rabbit antiserum (1/1000), washed three times in PBS/4% Tween 20 and incubated for 1 h with HRP goat anti rabbit IgG (1:20 000 dilution; Southern Biotechnology). HRP detection was performed as described above.

Figure 1 .

 1 Figure 1. Restriction map of the human pT § gene

Figure 2 .

 2 Figure 2. Genomic and protein organization of murine and human pT §

Figure 3 .

 3 Figure 3. Expression of two different pT § splice variants. pT § expression was analyzed by RT-PCR in human thymus and three different human acute lymphoblastic cell lines: HBL-all, MOLT4, and Jurkat. Specific transcripts were not found in Jurkat cells. Two different bands could be observed: one of the expected size of ˚640 bp, and a smaller one of ˚320 bp. The control RT-PCR for g -actin is also shown below.

Figure 4 .

 4 Figure 4. Immunoprecipitation of pT § 1 in the human acute lymphoblastic cell lines HBL-all and Jurkat.

Figure 5 .

 5 Figure 5. F23.1 (anti-V g 8 monoclonal antibody) immunoprecipitates of SCB29 cell lysates analyzed by nonreducing/ reducing SDS-PAGE. (A) Blots from surface biotinylated cells. 1: pT § 1, 2: TCR g (pT § 1 associated); 3: a 20-kDa covalently TCR g -linked protein, which may represent pT § 2; 4: TCR g (associated with the 20-kDa, TCR g -linked protein). No other covalently TCR g -linked protein could be detected. (B)The blot shown in (A) was destained and probed with the polyclonal rabbit anti-murine pT § antiserum R225. Spots 1 and 3 correspond to spots 1 and 3 seen in blot A, representing pT § 1 and the putative pT § 2, respectively. An additional spot could be detected by the pT § -specific antibody. As this protein (spot number 5) could not be detected by cell surface protein biotinylation, it probably represents an intermediate, intracellular pT § protein with incomplete posttranscriptional modifications.

Eur. J.Immunol. 1998. 28: 3824-3831 Genomic structure and expression of pre-T §

Acknowledgments: Oskar Lechner is a recipient of a Marie Curie Training Grant from the European Comission. Harald von Boehmer is supported by the Institut Universitaire de France, by the Juvenile Diabetes Foundation (USA), and by the Koerber Foundation (Germany). This work was supported in part by the Institut National de la Sant é et de la Recherche M édicale, Paris, and by the Facult é Necker Enfants Malades, Descartes Universit é, Paris.


Paola Del Porto

Ludovica Brunot

Marie-Genevieve Matreit

Harald Von Boehmer

Claude Saint-Ruf

Philippa Marrack

Cloning and comparative analysis of the human pre-T-cell receptor a-chain gene

In immature T cells the T-cell receptor (TCR) 18-chain gene is rearranged and expressed before the TCR a-chain gene. At this stage TCR 18 chain can form disulfide-linked heterodimers with the pre-T-cell receptor a chain (pTa). Using the recently isolated murine pTa cDNA as a probe, we have isolated the human pTa cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTa with the mouse pTa molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTa gene is expressed in immature but not mature T cells and is located at the p2l.2-pl2 region of the short arm of chromosome 6.

Intrathymic T-cell development is accompanied by ordered rearrangement of T-cell receptor (TCR) genes as well as by changes in expression of surface markers (1). At a certain developmental stage, CD4-, CD8-, CD3l0w, CD44+, CD25+ T-cell precursors downregulate CD44 expression and begin to rearrange the TCR (3-chain (TCRI3) locus. After productive TCR(3 genes are formed, thymocytes become CD25-, acquire CD4/CD8 coreceptors on the cell surface, and rearrange the TCRa locus (2)[START_REF] Pearse | Proc. Natl. Acad. Sci. USA[END_REF][START_REF] Snodgrass | [END_REF](5). At the CD4+ CD8+ TCRaf+ stage, thymocytes are subjected to positive and negative selection depending on the quality of TCR-major histocompatibility complex ligand interaction (6, 7). While the TCRao3 controls late thymic development, early developmental steps are con- trolled by the pre-TCR composed of the variant TCR,3 chain that is disulfide linked to the invariant pre-TCRa (pTa) protein recently described in mice (7-9). The TCRf3-pTa heterodimer is associated with CD3 molecules (10) and signals triggered by the pre-TCR induce expansion and differentiation of immature precursor cells (8,9,11). Here we report on the isolation and characterization of the human pTa cDNA and its comparison with the murine homologue.1 In addition we provide data on the chromosomal location as well as on pTa expression in thymic subsets.

MATERIALS AND METHODS

cDNA Isolation and Sequencing. A 3-year-old human thy- mus cDNA library constructed in AgtlO vector (1 x 106 clones) (Clontech; HL1 127A) was screened with a probe correspond- ing to the full-length murine pTa. Prehybridization and hy- bridization were done at 65°C in a solution containing 1 M NaCl, 50 mM Tris HCl (pH 7.2), 10% dextran sulfate, 1% SDS,

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and 0.250 mg of salmon sperm DNA per ml. Filters were washed in 2x SSC first at room temperature and then at 65°C. The murine cDNA was used as a probe and labeled with [a-32P]dCTP by random priming (DNA labeling kit; Boeh- ringer Mannheim). Inserts from positive clones were sub- cloned in the EcoRI site of pBluescript SK(-) vector. Plasmid DNA was purified according to Birnboim and Doly (12) for double strand and according to Sambrook (13) for single strand. Dideoxynucleotide sequencing was carried out either on single-stranded template or on double-stranded template obtained from the pBluescript sub'clone. Reading ambiguities were resolved by substitution of 7-deaza-dGTP and 7-deaza- dATP for dGTP and dATP, respectively, in sequencing reac- tion mixtures. Sequenase from United States Biochemical and T7 DNA polymerase from Pharmacia Biotech have been used.

DNA sequence analysis was done with the GCG program (Genetics Computer Group, program manual for the GCG package, version 7, April 1991). Homology searches in the EMBL (release 42.0, March 1995), GenBank (release 88.0, April 1995), and Swiss-Prot (release 29.0, June 1994) data banks were done with the FASTA [START_REF] Pearson | Proc. Natl. Acad. Sci[END_REF] program. Prediction of the secondary structure and hydrophilicity searches were done with the PEPTIDESTRUC=TRE program.

Cloning of the 5' and 3' Ends. Poly(A)+ RNAs were extracted from 3-month-old human thymus by using the citric acid method [START_REF] Schibler | [END_REF]. cDNA synthesis was performed on 1 ,ug of poly(A)+ RNA with an oligo(dT) adaptor 5'-GACTCGAG- TCGACATCGAT17-3' according to the supplier's recommen- dations (cDNA cycle kit; Invitrogen). To obtain the 3' end, a primer specific for the human pTa sequence (5'-CCTGCCT-TCTGAGGAGCTG-3') as deduced from analysis of the pos- itive AgtlO recombinant clone and the oligo(dT) adaptor were used for PCR. Thirty cycles of amplification (94°C for 40 sec, 50°C for 2 min, and 72°C for 3 min) were carried out followed by a 10-min final extension at 72°C in 100 ptl of PCR mixture containing 2 ,ul of cDNA and 50 pmol of each primer. A second round of amplification was performed on amplified material after size purification. The primers used for the second amplifi- cation were a nested pTa-specific primer (5'-TGGGGCTGAG-GGTCACAGCA-3') and the adaptor (5'-GACrCGAGTCGA-CATCGATT-3'). The amplified material was migrated on a 1% agarose gel, purified by adsorption on glass beads, and ligated into the pCR-Script SK vector (Stratagene).

For the 5' end amplification, the cDNA was synthesized using the pTa-specific primer 5'-GCCTCTCCTGACAGAT-GCAT-3'. The dG-tailing was performed in a final vol of 50 ,ul of tailing buffer with 15 units of terminal deoxynucleotidyltransferase (GIBCO/BRL). The first amplification of dG- Abbreviations: TCR, T-cell receptor; TCRf3 and -a, TCR 13 chain and TCR a chain; pTa, pre-TCRa; PKC, protein kinase C; C, constant; V, variable. §To whom reprint requests should be addressed. 1The sequence reported in this paper has been deposited in the GenBank data base (accession no. U36759).

12106 Immunology: Del Porto et al.

tailed cDNA was performed in 100 ,ul of PCR buffer using as primers an anchor poly(C) primer ( 16) and a human pTa-specific primer (5'-GGGAGAGATGGGCCAAGTTG-3'). Amplification was done for 25 cycles at 94°C for 1 min, 55°C for 1.5 min, and 72°C for 2.5 min. A second round of amplification was performed on size-selected amplified material with a nested pTa-specific primer (5'-TGATTGGTGGGGCCAGAGAAG-3') and the adaptor primer (16). The PCR product was cloned as described above.

Southern Blot. A Zoo-Blot from Clontech was hybridized either with the entire human pTa cDNA, with a 300-bp Pst I insert coding for the human pTa cytoplasmic tail, or with a PCR product corresponding to the murine cytoplasmic se- quence. Prehybridization and hybridization were done at 65°C as described. Membranes were washed in 2x SSC/0.1% SDS at room temperature and then at 65°C. Stringent washes were performed in 0.5 x SSC/0.1% SDS at 650C when the Zoo-Blot was hybridized with the entire probe. Probes were removed in 0.5% SDS at 90-1000C.

Monoclonal Antibodies. The following monoclonal antibod- ies were used in this study: phycoerythrin-coupled Leu-3a (anti-CD4 antibody; Becton Dickinson), fluorescein isothio- cyanate (FITC)-labeled Leu-4 (anti-CD3 antibody; Becton Dickinson), biotinylated Leu-2a (anti-CD8 antibody; Becton Dickinson).

Surface Staining and Cell Sorting. Cells were suspended in PBS containing 2% fetal calf serum (FCS) at 107 cells per ml and incubated with the desired antibodies at optimal concen- tration for 10-20 min on ice. After incubation, cells were washed twice in PBS plus 2% FCS and resuspended either in the same solution (in the case of direct staining with phyco- erythrin-conjugated, FITC-conjugated monoclonal antibod- ies) or (in the case of indirect staining with biotinylated antibodies) in optimally diluted APC (Southern Biotechnology Associates) and incubated for an additional 10-20 min on ice. Cell suspensions were filtered and sorted on FACStarPLUs (Becton Dickinson) instruments. Sorted populations were reanalyzed to test their purity, which was always found to be >99%.

Reverse Transcription PCR. Cells (5 x 104) were directly sorted into 500 ,tl of RNAzol (Cinna/Biotecx Laboratories, Friendswood, TX); nonsorted cells were washed in PBS and resuspended in RNAzol. Total RNA was extracted according to the manufacturer's protocol. cDNA was prepared with random hexamer primers and reverse transcribed with a Superscript kit (GIBCO/BRL). For PCR, primers used were oligonucleotides recognizing sequences in the 5' and 3' regions of the pTa (5'-GGCACACCCTTTCCTTCTCTG-3' and 5'- GCAGGTCCTGGCTGTAGAAGC-3') and actin (5'-ACA- CTGTGCCCATCTACGAGGGG-3' and 5'-ATCATGGAG- TTGAAGGTAGT TTCGTGGAT-3') cDNAs. PCRs were done in 30 jil of reaction mixture containing 50 mM KCl, 10 mM Tris HCl (pH 8.3), 10 mM mixed dNTP, 10 pM each oligonucleotide primer, and 1 unit of AmpliTaq DNA poly- merase (Roche Diagnostics). DNA was amplified for 35 cycles at an annealing temperature of 55°C with a thermal cycling machine (Perkin-Elmer/Cetus). A 9-,lI portion of each am- plified product was separated throughout a 1% agarose gel by electrophoresis and stained with ethidium bromide.

Chromosomal Localization by in Situ Hybridization. In situ hybridization was carried out on chromosome spread prepa- rations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 hr. 5-Bromodeoxyuridine was added for the final 7 hr of culture (60 ,ug per ml of medium) to ensure a posthybridization chromosomal banding of good quality. The pTa clone containing an insert of 800 bp in pBluescript was 3H labeled by nick-translation to a specific activity of 1.4 x 10 dpm ,ug-1. The radiolabeled probe was hybridized to metaphase spreads at a final concentration of 25 ng per ml of hybridization solution as described (17).

After coating with nuclear track emulsion (Kodak NTB2), the slides were exposed for 21 days at +4°C and then devel- oped. To avoid any slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered Giemsa solution and metaphase spreads were photographed. R-banding was then performed by the fluoro- chrome/phytolysis/Giemsa method and metaphase spreads were rephotographed before analysis.

RESULTS

Isolation and Analysis of Human pTa cDNA. Screening of a human thymus cDNA library led to the isolation of three positive clones, which were subcloned into pBluescript SK(-) and sequenced. Sequence analysis of a 931-bp insert from the longest clone (F6) revealed the presence of a long open reading frame. To obtain the complete sequence of the human pTa and to confirm it independently, a rapid amplification of cDNA ends (RACE) [START_REF] Frohman | Proc. Natl. Acad. Sci[END_REF] was undertaken with primers lo- cated in the immunoglobulin-like domain. Sequencing of 3' RACE products allowed the identification of 104 additional nt with one putative polyadenylylation signal 20 bp upstream of the poly(A) tail. Sequencing of the 5' products identified 60 additional nt in the 5' untranslated region. By the combination of these techniques a sequence of 1097 nt was obtained. These data are in agreement with the 1.1-kb band observed in Northern blots of human thymus mRNA hybridized with mouse or human pTa probe (data not shown). The included open reading frame contained 846 nt encoding a 282-aa protein including the N-terminal methionine (Fig. 1). The calculated molecular weight of the protein was 27,793. The overall identity with the murine pTa was 63%. Positionwise, the human ATG that heads a long open reading frame corresponds to the second in-frame ATG in the murine pTa sequence, suggesting that in the mouse the second ATG in the 5' region represents the initiator codon.

The deduced human amino acid sequence reveals a 5' hydrophobic region of 16 aa with 75% identity at the nucleotide level and 70% identity at the amino acid level with the murine pTa leader sequence. The following 130 aa show 83% identity with three cysteines at the conserved positions 31, 91, and 119. The putative extracellular region presents features of an immunoglobulin-like structural constant (C) domain as defined by Williams and Barclay [START_REF] Williams | [END_REF]. The sequence encom- passing aa 1-116 shows the characteristic seven ,B-strands, which could fold in two 13-sheets stabilized by an intrachain disulfide bond formed by the cysteines at positions 31 and 91 and a tryptophan in position 46 that is involved in establishing the tertiary structure (20). This structural organization is very well conserved between mouse and human. While the murine extracellular domain exhibits two potential sites of glycosylation, the human sequence contains only one. The conserved cysteine at position 119 is assumed to participate in formation of the interchain disulfide bond with TCR,B and is included in a small sequence of 15 aa that form the connecting peptide, which shows only 60% identity to the murine sequence. The following 20 aa share 80% identity to the murine transmem- brane region. They are mostly hydrophobic and the region contains two polar residues (arginine and lysine) that are separated by the same 4 aa observed in the murine sequence. This feature is also conserved in human and murine TCRa chains and is assumed to be essential for the assembly and transport of the TCRaf3-CD3 complex (21).

The homology between human and murine pTa ends abruptly 5 aa after the predicted transmembrane region. Furthermore, the length of the human pTa cytoplasmic tail (114 aa) differs from that of the mouse (31 aa). There are three potential protein kinase C (PKC) sites in this part of the human pTa as opposed to two in the mouse, which correspond positionwise to the two first PKC sites in the human pTa.

m MARTWLLLLLGVRCQALPSGIAGTPFPSLAPPITLLVDGRQHMLVVCLVLDAAPPGLDNPVWFSAGNGSAL h --G---------G-P---T-VG-----------M-----K-Q-V-------V------S-I---------

- D L F a 56 v -CHO- v m DAFTYGPSLAPDGTWTSLAQLSLPSEELEAWEPLVCHTRPGAGGQNMSTHPLQLSGESSTARSCFPEPLGG h ----------T--------H--------AS--------G---E-HS-.--Q-MH----A----T-PQ---R- 127 +-M-+ -PKC--PKC- m TQRQVLWLSLLRLLLFKLLLLDVLLTCSHLRLHVLAGQHLQPPPSRKSLPPTHRIWT* h -PGGA---GV------------L-----C-CDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRP -PKC- -PKC- -PKC- 198 h QPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSRLRAPSSSLGAFFRGDLPPPLQAGAA* FIG. 1.
Comparison of the deduced amino acid sequence encoding the human and mouse pTa cDNA. Human sequence (h) is aligned with the previously described murine pTa sequence (m) (accession no. U16958). Amino acid residue numbers start with -16 as the first position of the leader and proceed from +1 of the putative mature proteins; locations of the potential N-linked glycosylation site (CHO), the predicted leader (peptide signal), the transmembrane (TM), and the potential PKC phosphorylation sites are indicated. The three cysteines at positions 31, 91, and 119 are marked (v). The conserved basic amino acids arginine and lysine, located in the transmembrane region, are indicated (+) in the lower line. The conserved position of the ,3-strands (A, B, C, D, E, F, and G) deduced by computer analysis of the secondary structure of the murine and the human sequence are shown above the two sequences. Hyphens replace residues in the human pTa that are identical to the corresponding residues in mouse pTa.

Unlike the murine pTa, the human sequence does not contain a PPGHR CD2-like motif, which is believed to play a role in CD2-mediated T-cell activation (22). Except for a proline triplet at the 3' end, the human pTa cytoplasmic tail does not contain a proline-rich region that could constitute an SH3 domain binding region (23), as found in the murine pTa.

Interspecies pTa Hybridization. To determine whether nucleotide sequence homologies could be detected by cross- hybridization at low stringency, we performed Southern blot analysis of EcoRI-digested DNA from several species. When the entire human pTa cDNA was used as a probe at least one hybridizing band was detected in all species analyzed. The same result was obtained with a probe corresponding to the human extracellular immunoglobulin-like domain only. In contrast, with probes corresponding to the cytoplasmic tails of either human or murine pTa, hybridization was obtained only with human and monkey DNAs or murine and rat DNAs, respectively (Fig. 2). Thus, among different species, the extra- cellular immunoglobulin-like domain appears much better conserved than the cytoplasmic tail, a result consistent with the sequence data of human and murine pTa.

Human pTa Chromosomal Location. In 100 metaphase cells examined after in situ hybridization, there were 214 silver grains associated with chromosomes and 58 of these (27.1%) were located on chromosome 6; their distribution on chromo- some 6 was not random: 44 of the 58 silver grains (76%) were localized on the p2l.2-p12 region of the short arm of chro- A mosome 6 (Fig. 3). Thus, the human pTa gene maps to the 6p21.2-6pl2 region of the human genome.

Recently, the murine pTa gene was localized in the D.E1 region of chromosome 17 (24), which is homologous to the short arm of human chromosome 6. Thus, in both human and mouse the pTa gene is localized in the area of the major histocompatibility complex.

Intrathymic Expression of Human pTa. Human pTa RNA was detected by PCR analysis using the primers described. Human thymocytes were separated into immature CD4- CD8-, CD3I0W, CD4+ CD8-, CD3I0w, CD4+ CD8+, CD3+ as well as mature CD4+ CD8-CD33+, CD4-CD8+ CD3++, and CD4-CD8-CD3++ thymocytes (25, 33). As shown in Fig. 4, pTa RNA is detected in the immature but not the mature subsets, and its expression is strongest in the CD4+ CD8-CD3l0w fraction of cells that are on their way to differentiate into CD4+ CD8+ thymocytes.

DISCUSSION

In this paper, we report the nucleotide sequence of the human pTa cDNA. Analysis of the human and murine pTa cDNA shows the existence of conserved and nonconserved regions in the protein that may be related to its function. Alignment of the extracellular regions of the human and mouse pTa shows that they are identical in length. The highest degree of homology is detected in the peptidic stretch corresponding to the immunoglobulin-like C domain (20), where 83% of identity Interspecies Southern blot analysis. EcoRI-digested DNA from human (lanes 1), monkey (lanes 2), rat (lanes 3), mouse (lanes 4), dog (lanes 5), and cow (lanes 6) was analyzed by Southern blotting. The blot was successively hybridized with probes corresponding to the entire human pTa cDNA (A) and part of the cDNA encoding the cytoplasmic tails of human (B) and mouse (C) pTa. is observed. This score is greater than the identity found between the human and murine C regions of TCRa ("60%). This remarkable conservation in the extracellular region of pTa could reflect the constraints imposed by the interaction with a conserved thymic (or extrathymic) ligand, which could promote expansion and differentiation of the T-cell lineage through signal transduction by the pre-TCR. Alternatively, the conservation could be due to pairing with the TCR,B protein. Some of.the conservation could also be imposed because, as in pre-B cells, an additional variable (V) domain in pre-T cells might be noncovalently associated with the immunoglobulinlike domain of pTa. This could be compatible with the notion that the TCRl3-pTa heterodimer is inefficiently transported to the cell surface in a mature T-cell line (9). On the other hand, there are several pieces of evidence that suggest that the structure of TCRf3 differs from that of the immunoglobulin heavy chain. Vp and C, are not structurally autonomous units (26) unlike the V and the C regions of immunoglobulin heavy chain (27). This is confirmed by analysis of the three- dimensional structure of TCRI3 (28), showing that the V, and C, domains are in intimate contact in the crystal structure. The Vp is folded on the C,3 domain in a rigid conformation, indicating that the interaction between the V regions of the a and ,3 chains could be reduced. Because of the stability of TCRJ3 by itself, there may be no requirement for an associated V pre-T-cell region in the pre-TCR. Furthermore, it has been shown that expression of a mutilated V,3 gene permits the intrathymic double-negative to double-positive transition to occur (29). This could suggest that the V domain of TCR13 is not an essential part of the pre-TCR complex to achieve signal transduction and, therefore, that interaction of a V pTa I + + + + C t 00 C0 t co C0 -0 00 0 00 0in0 0 0 0 0 0 0 0 0 0 0 0 0 0 000 00 0 domain with the Vp domain, analogous to the binding of V pre-B cell to V,1 in pre-B cells, is not needed to promote differentiation. These considerations are at least consistent with the idea that the pTa-TCRI3 complex could be sufficient to drive thymocyte development without the association of a V-like domain.

The peptide connecting the pTa transmembrane region with the immunoglobulin-like domain shows less homology (60%) but contains a conserved cysteine in position 119, expected to participate in the disulfide bond with TCRf. A high degree of conservation is observed in the transmembrane region of pTa, which contains the basic amino acids arginine and lysine that are also found in TCRa and TCR8 and appear necessary for interaction with the CD3 molecules (21). These data indicate that the human pTa molecule shares with its mouse counter- part all the features necessary to form a complex with TCR,B and the CD3 complex. It has been reported (10) that a weak association exists between the pre-TCR ap and the ; chains of the CD3 complex. A highly conserved phenylalanine at posi- tion 195 of the TCRa C region has been shown to be essential for a high avidity interaction with the ; chains (30,31). The absence of this phenylalanine residue in the corresponding part of the extracellular regions of human and murine pTa could account for the poor association of pre-TCR aj3 chain with the r chain.

The homology between hum-an and mouse pTa breaks off abruptly at the end of the transmembrane region. In fact, the 127 aa that are predicted to compose the human cytoplasmic region do not show any significant identity with the 30 aa residues found in the mouse pTa cytoplasmic tail. In this region of the human molecule, in addition to the presence of potential PKC sites present in the two molecules, we could identify neither a CD2-like motif (PPGHR) ( 22) nor proline- rich regions potentially involved in SH3 binding (23).

These results argue against the previously discussed hypoth- esis (9) that the cytoplasmic part of pTa could play a role in signal transduction of the pre-TCR through binding with proteins bearing SH3 domains like p56Ick. Indeed, the fact that pre-TCR is associated with the signal transduction CD3 mol- ecules and that the phosphorylation pattern of T-cell activation is very similar after pre-TCR or TCRaB crosslinking (unpublished observations) could suggest that the cytoplasmic pTa tail has no special role in signal transduction.

The lack of identity between the human and murine cyto- plasmic tails could reflect a divergence in gene structure. In contrast to the human sequence, the murine pTa sequence contains a B2 repetitive element in the 3' untranslated region that provides the polyadenylylation site. This type of element is known to generate recombination or translocation events by insertional mutagenesis. Moreover, a difference between the gene structure of the murine pTa gene and the TCRa and TCR6 genes is that the pTa transmembrane region, the cytoplasmic tail, and the 3' untranslated region are encoded by a single exon (24), whereas in TCRa and TCR8 genes the 3' Semiquantitative analysis of pTa expression on human thymocyte subsets. PCR amplifications of pTa-and actin-specific sequences were performed on serial dilutions of cDNAs. Amplified material was separated by agarose gel electrophoresis and stained with ethidium bromide. Phenotypes of the thymic populations are indicated. untranslated region is encoded by a separate exon. These differences could suggest that the 3' part of the pTa molecule has been subjected to variation. Analysis of the genomic organization of the human pTa should help to define whether differential splicing sites exist between human and murine pTa.

The Zoo-Blot analysis indicates again that a portion of the extracellular part of pTa is conserved in all the species analyzed and is compatible with the idea that some type of selective pressure may have conserved the extracellular do- main of the pTa but not the cytoplasmic region.

Overall, the human and murine pTa sequences for both the extracellular and cytoplasmic regions show no similarities with any published sequences in the data banks. We did not find any evidence for the existence of a pTa pseudogene when screen- ing the library or in Southern blot analysis. Thus, unlike the A5 gene (32), the pTa gene may not belong to a cluster of similar genes.

The chromosomal location of pTa on the short arm of human chromosome 6 was expected after it was shown that the murine pTa gene is located in the D/E1 region of chromosome 17. In fact, these regions are syntenic and harbor the major histocompatibility complex locus.

Finally, the expression pattern of pTa in the human thymus corresponds to that previously observed in the murine thymus. Taking into consideration the differences in thymocyte surface markers in the two species, our data indicate that in both human and mouse pTa expression is developmentally regu- lated.

  Proc. Natl. Acad. Sci. USA 92(1995) Proc. Natl.Acad. Sci. USA 92 (1995

  FIG.2. Interspecies Southern blot analysis. EcoRI-digested DNA from human (lanes 1), monkey (lanes 2), rat (lanes 3), mouse (lanes 4), dog (lanes 5), and cow (lanes 6) was analyzed by Southern blotting. The blot was successively hybridized with probes corresponding to the entire human pTa cDNA (A) and part of the cDNA encoding the cytoplasmic tails of human (B) and mouse (C) pTa.

  Immunology: Del Porto et al. 12108 Immunology: Del Porto et al.

FIG. 3 .

 3 FIG.3. Idiogram of the human G-banded chromosome 6 illustrat- ing distribution of the labeled sites with the human pTa probe.

  FIG.4. Semiquantitative analysis of pTa expression on human thymocyte subsets. PCR amplifications of pTa-and actin-specific sequences were performed on serial dilutions of cDNAs. Amplified material was separated by agarose gel electrophoresis and stained with ethidium bromide. Phenotypes of the thymic populations are indicated.

Proc. Natl. Acad. Sci. USA 92(1995) Proc. Natl. Acad. Sci. USA 92 (1995) 12109

We thank M. Reth (Freiburg) for helpful advice concerning analysis of the pTa sequence. The Basel Institute for Immunology is supported by F. Hoffmann-La Roche Ltd, Basel. This work was also supported by Institut National de la Sante et de la Recherche Scientifique, Unite 373. H.v.B. is supported by the Human Frontier Science Program Organization. P.D.P. is a European Molecular Biology Organization postdoctoral fellow.