Biomedical subjects
Publications and source records attributed to C Benoist.
RJ 2.2.5 is an immunoselected mutant of the Burkitt lymphoma line Raji. It fails to display MHC class II antigens at the cell surface due to a transcriptional defect. We have identified the function of a regulatory factor that is defective in RJ 2.2.5 cells; this factor is absolutely required for the activity of an MHC class II gene enhancer.
A set of transgenic mouse lines carrying Ek alpha genes with promoter region deletions was created in an attempt to compartmentalize MHC class II gene expression. Fine immunohistological analyses established that one transgenic line is essentially devoid of E complex in the thymic cortex, another displays almost no E in the thymic medulla or on peripheral macrophages, and two lines display no E on greater than 98% of B cells. We have assayed these mice for immune function: E-dependent tolerance, antigen presentation, T cell priming, and antibody response. Certain of the findings are difficult to reconcile with currently popular hypotheses, e.g., tolerance induction to E molecules in the virtual absence of E complex in the thymic medulla and efficient antibody responses to E-restricted antigens when almost all B cells are E-.
The E alpha class II gene of the major histocompatibility complex is expressed in a variety of immunocompetent cells. Part of the control of tissue-specific expression is mediated by a block of sequences found far upstream of the transcriptional startsite; this stretch is necessary for expression in the B lymphocytes of transgenic mice, but largely dispensable elsewhere. We review the evidence for the role of this region in E alpha transcription in transgenic animals, as well as data from transfections into tissue-culture cells, which indicate that this region has non-specific enhancer activity. We discuss possible models to explain how a non-specific enhancer can participate in cell-specific control.
Transcription of major histocompatibility complex class II genes is elaborately regulated. Mouse class II genes are transcribed primarily in B cells, peripheral macrophages and interdigitating cells, and thymic cortical and medullary cells. In this study, we began to identify the DNA sequences and protein factors that control expression of a class II gene in B cells, addressing in particular how closely they resemble those that regulate immunoglobulin gene expression. We describe a region upstream of the E alpha gene that is crucial for its transcription in the B cells of transgenic mice but is less important in cultured B-cell lines. The sequence of this region reveals several familiar motifs, including a second X-Y pair reminiscent of that residing in the promoter-proximal region of all class II genes, a B motif strikingly homologous to that associated with the immunoglobulin kappa gene enhancer, several Ephrussi motifs, and a Pu box-like sequence very similar to that implicated in simian virus 40 and lymphotrophic papovavirus expression in B cells. Careful study of the proteins that bind specifically to these different motifs prompts us to suggest that major histocompatibility complex class II and immunoglobulin genes rely on quite different factors to achieve B-cell-specific expression.
NF-Y is a sequence-specific DNA-binding protein that interacts with the conserved Y motif of the major histocompatibility complex class II gene, E alpha. Since it is actually a CCAAT box-binding protein, NF-Y also attaches to other promoters bearing CCAAT sequences; yet, it is neither of the previously described transcription factors, CBP or CTF/NF-1. In this report, we document the cell-type distribution and various biochemical properties of NF-Y. The most important findings are that this protein is ubiquitously distributed, that it is probably a metallo-protein, that it has a protease-resistant DNA-binding domain and that the NF-Y/E alpha-olgo complex seems extremely large (greater than 200kD). These data should prove useful in comparisons of NF-Y with other sequence-specific DNA-binding proteins; they have already provided new insights into NF-Y's structure.
NF-Y is a sequence-specific DNA-binding protein that recognizes the Y box, a promoter element common to all major histocompatibility complex class II genes. Since the 14-base Y element harbors a CCAAT box in reverse, we were prompted to ask whether NF-Y is actually a CCAAT box-binding protein and whether it is related to the previously described CCAAT-binding factors CBP and CTF/NF-I. Data from gel retardation, methylation interference, saturation mutagenesis, and cross-competition experiments establish definitively that NF-Y is an entirely distinct CCAAT box-binding entity. Moreover, these experiments have uncovered a fourth CCAAT-binding protein, NF-Y(star) that interacts with the thymidine kinase promoter. Clearly, then, there exists a multiplicity of factors that recognize CCAAT sequences; it now becomes imperative to understand the functional significance of this multiplicity.
A conserved sequence motif exists at the 5' end of all major histocompatibility complex class II genes. This motif consists of the 14-base X and Y boxes separated by a short stretch of variable sequence. In this report, we provide evidence that the X and Y boxes play an important role in controlling transcription of the murine class II gene E kappa alpha. We have developed transgenic mouse lines that carry E alpha genes cleanly deleted for either the X or Y box and have compared the expression of these mutant transgenes with that of a nondeleted control. Both the X and Y segments appear critical for accurate and efficient transcription of E kappa alpha. The most drastic effect is seen with gamma-interferon-treated macrophages, where deletion of the Y box completely abrogates transcription initiated by the normal promoter. In addition, we identify proteins from nuclear extracts that bind specifically to the X or Y box.
Ia antigens are polymorphic cell-surface molecules that control the immune response. We have begun to localize important functional sites on one of the Ia molecules, A alpha. Herein, we focus on the A alpha k and A alpha b alleles and ask what defines "b-ness" and "k-ness" for a panel of monoclonal antibodies. Two independent experimental strategies are employed: the ability of 12 monoclonal antibodies to recognize L cell transfectants bearing chimeric and mutant A alpha chains is assessed, and the amino acid sequences of A alpha chains expressed by immunoselected B lymphoma mutants are deduced. For each antibody, we identify a stretch of polymorphic residues critical for recognition; for several, we can pinpoint a single amino acid. Certain stretches of A alpha (depending on the allele) appear strikingly immunodominant.
H-2u haplotype mice are unique among all E alpha+ strains because they do not provide in heterozygotes an E alpha chain that interacts with E betak,s,etc. sufficiently well to allow certain E-restricted immune responses. As a first step in understanding this peculiarity, we have sequenced E alpha u and E beta u cDNA and compared the derived amino acid sequences with those of previously analyzed alleles. Although no glaring structural abnormalities were found, we have identified some u-specific residues and suggest which are the most likely to provoke a pairing anomaly.
One of the more intriguing puzzles in immunology is the genetic basis for control of murine T-cell I-J determinants. Molecules bearing I-J determinants (I-J molecules) play a role in information trafficking among immunocompetent cells, probably serving as self-recognition molecules that channel regulatory factors to their appropriate target cells. Although it is clear that I-J polymorphism is influenced by the major histocompatibility complex (MHC), molecular genetic studies provide evidence that an MHC gene does not encode I-J molecules. A possible explanation for this paradox is that I-J molecules are a set of non-MHC-encoded T cell receptors that are directly or indirectly selected for by self-MHC products. One key to resolving the genetic and molecular basis for control of I-J determinants is the identification of the MHC gene(s) involved. Herein, data are presented which show that E alpha transgenic mice express an altered I-J phenotype, providing clear evidence that I region class II genes influence I-J polymorphism. Although further study is required to resolve how class II genes mediate this effect, this is a major piece to the I-J puzzle.
The murine Ia antigens, heterodimeric glycoproteins on the surface of immunocompetent cells, restrict immune recognition by their influence on cell-cell interactions. Many serological specificities have been mapped to these molecules, and monoclonal antibodies directed against some of these determinants block antigen presentation to T lymphocytes. As a step toward a better understanding of Ia function, we sought to define the molecular basis of Ia.2 and Ia.19, specificities found on the A alpha chain of only the k or of both k and r haplotypes, respectively. We report nucleotide sequences for the A alpha chain cDNAs of the k, r, and s haplotypes, which, when compared to previously published A alpha sequences, demonstrate the existence of one k-specific amino acid residue and of another present only in the k and r haplotypes. These residues must thus play an important role in the generation of Ia.2 and Ia.19 specificities.
Class II major histocompatibility complex (MHC) molecules, the Ia antigens, are intimately involved in regulating the intensity and specificity of the cellular and humoral responses to T cell-dependent antigens. One approach to understanding the mechanism of this regulation is to analyze the structure and allelic polymorphism of Ia molecules. In addition there are regulatory polymorphisms in the expression of the I-E alpha and I-E beta class II MHC polypeptide chains. Analysis of the cDNA sequence indicates that I-A and I-E alpha chains are similar with short stretches of homology and other regions of nonhomology. Analysis of Northern blots of mRNA indicates that at least three separate types of regulatory polymorphisms result in failure of expression of I-E alpha. Comparison of allelic sequences of six alleles of the I-A alpha chain shows that almost all of the allelic polymorphism is in the first domain and that within the first domain it is clustered in three allelic hypervariable regions within the first domain of I-A alpha. The structural and functional implications of these findings are discussed.
The murine Ia antigens, encoded by the I region of the major histocompatibility complex, are cell-surface glyco-proteins (consisting of alpha and beta polypeptides) thought to be involved in the control of immune responsiveness. Mice of haplotypes b, s, q, and f fail to express one of the Ia antigen complexes, the E complex, on the cell surface. We have attempted to determine at the molecular level how such a defect (or defects) might be generated. By using I-region E alpha and A alpha gene probes for analyses of RNA and DNA structure, it was possible to conclude that at least three mechanisms can operate. Mice of haplotypes b and s bear a deletion in the E alpha gene, f haplotype mice synthesize predominantly an E alpha mRNA of aberrant size, and mice of the q haplotype seem to have a defect in RNA processing or a problem with mRNA stability, or both.
To investigate the sequences necessary for proper initiation of transcription of SV40 early genes, we have constructed several deletion mutants in the promoter region. The TATA box region is apparently involved in fixing initiation precisely within a narrow area, but is dispensable for gene expression, while the sequences located more than 150 base pairs upstream are indispensable.
A recombinant plasmid has been constructed for the expression of inserted DNA sequences coding for polypeptide chains using the simian virus 40 early promoter and splicing and polyadenylylation signals from the rabbit beta-globin gene. The coding regions for two prokaryotic methotrexate-resistant dihydrofolate reductases were introduced into the expression vector. When mouse fibroblasts were exposed to these recombinant plasmids, it was possible to select methotrexate-resistant clones that had integrated the plasmids and produced a chimeric RNA coding for the prokaryotic enzyme.
We present the sequence of regions of the chicken ovalbumin gene believed to be important in the control of initiation of transcription, splicing, and transcription termination or polyadenylation. Comparison with corresponding areas of other genes reveals some homologous regions which might play a role in these processes.
A recombinant plasmid was constructed by insertion of the early genes of simian virus 40 (SV40) into pBR322. When it was introduced into eukaryotic cells, the SV40 early genes were expressed. We have made deletion mutants of this plasmid, from which the major cap sites of SV40 early mRNAs have been removed along with some of the sequences upstream. The deleted sequences appear to be dispensable for early gene expression, but this does not necessarily imply that they serve no function in the initiation of transcription on wild-type SV40.
We have determined the sequence of ovalbumin RNA (ov-mRNA) using a double-stranded cDNA (dscDNA) plasmid. We have also determined the sequence of the previously characterized exonic regions of the chicken ovalbumin gene. The comparison of these various sequences has shown that there are no additional interruptions in the mRNA-coding sequences above those 7 already characterized. There is only one single base discrepancy between the two mRNA sequences determined using the dscDNA or the genomic clones. This demonstrates the accuracy and reproducibility of the cloning and sequencing techniques. The ovalbumin mRNA sequence was found to be 1872 nucleotides in length, 13 nucleotides larger than the previous value reported by McReynolds et al. [Nature 273, 723-728 (1978)].