A Developmental Bias in Reading Frame Usage by Human Fetal Thymic TCRBDJ Transcripts is not Present in Genomic TCRBDJ Rearrangements
Developmental Immunology
A Developmental Bias in Reading Frame Usage by Human Fetal Thymic TCRBDJ Transcripts is not Present in Genomic TCRBDJ Rearrangements
JAMES F. GEORGE
YASUSHI MATSUURA
JACQUELYN A. BYRNE
EUGENE L. LIU
DENISE R. SHAW
JOHN F. KEARNEY
We have previously reported that reading-frame usage and functional diversification is developmentally regulated, with virtually all TCRB DJ mRNA transcripts using a single reading flame at 8 weeks of gestational age, tapering to 50% by adult life. We used the polymerase chain reaction to create genomic libraries of DJ rearrangements in the TCRB locus from thymuses at 7.7, 10, and 16 weeks of gestational age, and from adult thymuses. Clones were randomly picked and sequenced to determine junctional sequences and reading-flame utilization. The resulting data address the hypothesis that cells bearing genomic joints in reading flame one are preferentially selected during fetal life. This hypothesis predicts that reading-flame bias would also be observed among genomic DJ joints. Instead, we observed random utilization of the three possible D-region reading flames among genomic Dlsl -> Jlsl joints during fetal life. Similar results were obtained at 7.7 weeks of gestational age in a second thymus in which both RNA and DNA were simultaneously isolated and used to create libraries of TCRBDJ transcripts or rearrangements. We conclude that reading-flame utilization is random among genomic D s 1-JB rearrangements and that the preferential usage of a single reading flame among mRNA transcripts of TCRB DJ transcripts is the result of preferential transcription of genomic TCRB DJ joints in a single reading flame, or that TCRB DJ transcripts have a longer half-life than transcripts in reading flames two or three.
T-cell receptor; gene rearrangement; reading-frame usage
INTRODUCTION
In previous studies, we established a system for
examining the molecular events that occur during the
rearrangement and expression of the TCR[ locus
during early thymic development in humans. We found
that the expression of partial rearrangements (D ==>
J) in the human TCRI3 locus during fetal life is
regulated in a manner that suggests that there may be
selective events that occur prior to the expression of a
complete TCR chain on the cell surface. Of three
possible reading frames, one D-region reading frame
is predominantly used among DJ transcripts. At
8 weeks of gestation, 94% of the transcripts used
reading frame one, a proportion that dwindled to 67%
at 16 weeks and to 55% in the adult
(George and
Schroeder, 1992)
. Thus, the reading-frame bias is
most pronounced during the critical 7-14 week period
of gestation. Our hypothesis is that this bias in
reading-frame utlization is a function of selection of
thymocytes bearing rearrangements in reading frame
one. Here we demonstrate that genomic TCRB D => J
rearrangements exhibit random reading frame usage
in contrast to D => J transcripts, which are highly
biased in favor of a single reading-frame. Therefore,
we conclude that the observed bias in reading-frame
usage is not due to cellular selection, but is a result of
either selective transcription or a longer half-life of
DJ transcripts in reading frame one.
RESULTS
Structure and Composition of Genomic
DBIsI-JBlsl Rearrangements
A total of 127 unique genomic TCRB DJ clones were
sequenced from thymuses obtained at gestational ages
of 7.7 weeks (two thymuses, n 20 and n=21), 10
weeks (n=24), 16 weeks (n=22), and adult life (two
thymuses, n=22 and n=18). In accordance with our
previous analysis of TCRBDJ transcripts
(George and
Schroeder, 1992)
, the developmental control of
N-region addition also extends to genomic DJ joints.
At 8 weeks of gestational age, the mean proportion of
joints bearing N-region nucleotides was only 22%,
but increased to 50% by 16 weeks of gestational age,
reaching a maximum mean proportion of 83% during
adult life (Table I). This increase in the proportion of
genomic DJ joints bearing N-region nucleotides
showed a positive linear correlation with gestational
age through 16 weeks (r= 0.98: Y= 3.35x- 2.91).
Similarly, the mean number of N-region nucleotides
per genomic DJ joint also increased with time, with a
positive linear correlation with increasing gestational
age (r 0.99: Y 0.27x- 0.93). There were no
differences in the average number of nucleotides removed
from the genomic sequence of the gene segments
composing the DJ joints (Table I).
D-Region Reading-Frame Usage Is Random in
Genomic DJ Joints
Figure 1 shows reading-frame usage among 106
unique DJ joints isolated from human thymuses
obtained at gestational ages of 7.7, 10, 16 weeks, and
from two thymuses obtained from healthy adults.
Unlike fetal TCRB DJ mRNA transcripts, in which
the reading-frame usage is strongly biased toward the
use of a single reading-frame
(George and Schroeder,
1992)
genomic TCRB DJ joints exhibit random
reading-frame usage during fetal life. Surprisingly, the
proportion (...truncated)