Developmental and functional impairment of T cells in mice lacking CD3 zeta chains.

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.     

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

Evolution of human immunoglobulin kappa J region genes

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.     

Cloning and expression of the rat interleukin-3 gene.

Genomic clones carrying the rat interleukin-3 (IL-3) gene have been isolated and the nucleotide sequence of the gene determined. Alignment of this sequence with that of the mouse IL-3 gene has allowed the structure of the rat IL-3 gene to be deduced. The intron-exon boundaries are conserved and extensive nucleotide homology (approx 90%) is present in the 5' flanking region and the portion of the gene coding for the signal peptide. Several proposed regulatory sequences are conserved and an analogous element to the tandem repeat in intron 2 of the mouse gene is also present. The predicted amino acid sequence for mature rat IL-3 shows surprisingly low homology (54%) with its murine counterpart, although all four cysteine residues are conserved. The rat IL-3 gene was expressed in monkey COS-1 cells and colony assays established that rat IL-3 is a multi-lineage haemopoietic growth regulator. There was little cross-reactivity of the respective IL-3 species on mouse and rat bone marrow cells suggesting that rat IL-3, in concert with its receptor, has evolved significantly away from the mouse IL-3/receptor system.     

Cloning, genomic structure and chromosomal localization of the gene encoding mouse DNA helicase RecQ helicase protein-like 4

Five members of the RecQ helicase family, RECQL, WRN, BLM, RECQL4 and RECQL5 have been identified in humans. WRN and BLM have been demonstrated to be the responsible genes in Werner and Bloom syndromes, respectively. RECQL4 (RecQ helicase protein-like 4) was identified as a fourth member of the human RecQ helicase family bearing the helicase domain, and it was subsequently shown to be the responsible gene in Rothmund-Thomson syndrome. Here, we isolated mouse RECQL4 and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL4 consists of 3651 base pairs coding 1216 amino acid residues and shares 63.4% of identical and 85.8% of homologous amino acid sequences with human RECQL4. The RECQL4 gene was localized to mouse chromosome 15D3 distal-E1 and rat chromosome 7q34 proximal. They were mapped in the region where the conserved linkage homology has been identified between the two species. Twenty-two exons dispersed over 7 kilo base pairs and all of the acceptor and donor sites for splicing of each exon conformed to the GT/AG rule. Our observations regarding mouse RECQL4 gene will contribute to functional studies on the RECQL4 products.     

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.     

Relationship of viroids and certain other plant pathogenic nucleic acids to group I and II introns

Summary The nucleotide sequences of viroids contain features believed to be essential for the splicing of group I introns. Common sequence elements include a 16-nucleotide consensus sequence and three pairs of short sequences arranged in the same sequential order in both types of RNAs. The calculated probability of finding sequences resembling the 16-nucleotide consensus sequence in random nucleotide chains showed that at low fidelity (up to 5 mismatched nucleotides), the number of such sequences in viroids, plant viral satellite RNAs, plant viral RNAs and one plant viral DNA, group I introns and flanking exons does not significantly differ from the number expected at random. As the degree of fidelity is increased, the number in both introns and viroids, but not in exons or the other plant pathogens examined, greatly exceeds that expected in random chains. These findings suggest that viroids may have evolved from group I introns and/or that processing of viroid oligomers to monomers may have structural requirements similar to those of group I introns. The nucleotide sequences of viroids do not show close homology with two conserved regions of group II introns, the 14-base pair consensus region and the 5 terminal segment. However, close homology does exist between the conserved sequence of the 3 terminal segment of group II introns and viroids thus suggesting a possible evolutionary or functional relationship.     

Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.     

Identification of a novel gene on chromosome 7q31 that is interrupted by a translocation breakpoint in an autistic individual

The results of genetic linkage studies for autism have suggested that a susceptibility locus for the disease is located on the long arm of chromosome 7 (7q). An autistic individual carrying a translocation, t(7;13)(q31.3;q21), with the chromosome 7 breakpoint located in the region of 7q implicated by genetic studies was identified. A novel gene known as "RAY1" (or "FAM4A1") was found to be directly interrupted by the translocation breakpoint. The gene, which was found to be encoded by 16 exons with evidence of alternative splicing, spanned > or =220 kb of DNA at 7q31.3. Mutation screening of the entire coding region in a set of 27 unrelated autistic individuals failed to identify phenotype-specific variants, suggesting that coding region mutations are unlikely to be involved in the etiology of autism. Apparent homologues of RAY1 have also been identified in mouse, rat, pig, chicken, fruit fly, and nematode. The human and mouse genes share similar splicing patterns, and their predicted protein products are 98% identical.     

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into palsma cells. CD19 is a member of Ig gnee superfamily with two extreacellular Ig-like domains separated amino acid cytoplasmic domain. In this study, Southern blot analysis revelaed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned 8 kilobases (kb) of DNA in human and 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5 of the putative translation initiation AUG codon as well conserved in sequence between human and mouse and contained potential trasncription regulatory elements. In addition, the 3 untranslated regions (UT) of the CD19 genes following the termination codon were conservedf in sequence. The high level conservation of nucleotide sequences between species in all exons and 5 and 3 UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.The nucleotide sequence database reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: human CD19 gnee, M62544 to M62550; mouse CD19 gene, M62551 to M62553.     

T cell development in mice lacking the CD3-zeta/eta gene.

The CD3-zeta and CD3-eta polypeptides are two of the components of the T cell antigen receptor (TCR) which contribute to its efficient cell surface expression and account for part of its transducing capability. CD3-zeta and CD3-eta result from the alternative splicing of a single gene designated CD3-zeta/eta. To evaluate the role of these subunits during T cell development, we have produced mice with a disrupted CD3-zeta/eta gene. The analysis of thymocyte populations from the CD3-zeta/eta-/- homozygous mutant mice revealed that they have a profound reduction in the surface levels of TCR complexes and that the products of the CD3-zeta/eta gene appear to be needed for the efficient generation and/or survival of CD4+CD8+ thymocytes. Despite the almost total absence of mature single positive thymocytes, the lymph nodes from zeta/eta-/- mice were found to contain unusual CD4+CD8- and CD4-CD8+ single positive cells which were CD3-. In contrast to the situation observed in the thymus, the thymus-independent gut intraepithelial lymphocytes present in zeta/eta-/- mice do express TCR complexes on their surface and these are associated with Fc epsilon RI gamma homodimers. These results establish an essential role for the CD3-zeta/eta gene products during intrathymic T cell differentiation and further emphasize the difference between conventional T cells and thymus-independent gut intraepithelial lymphocytes.     

Mosaic evolution of prepropancreatic polypeptide. II. Structural conservation and divergence in pancreatic polypeptide gene

We demonstrated that nucleotide and amino acid sequences in the carboxyl-terminal regions of rat, mouse, and human prepropancreatic polypeptide exhibit a high degree of divergence, whereas the amino-terminal domains are highly conserved. To understand the molecular basis of this divergence and conservation, we determined the nucleotide sequence of the rat pancreatic polypeptide gene from an islet genomic library and compared it with that of the human gene. Exon 2 of the rat gene encodes the signal peptide and pancreatic polypeptide, exon 3 encodes the carboxyl-terminal region, and exons 1 and 4 encode the 5'- and 3'- untranslated regions of the mRNA, respectively. Exons 1 and 2 of rat and human genes are well conserved. The rat and human genes, however, have exons 3 and 4 of different lengths and heterologous nucleotide sequences. Mutational accumulation in exons 3 and 4 and intron 3 of the rat gene appears to have caused splice junction sliding and translational frameshift, resulting in a structural divergence in the carboxyl-terminal region. Available evidence indicates that the mosaicism of structural conservation and divergence in pancreatic polypeptide genes may have been caused by a difference in the evolutionary rates of the genomic regions.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

New mutant mouse with skeletal deformities caused by mutation in delta like 3 (Dll3) gene.

We have established a new mouse strain with vertebral deformities caused by an autosomal single recessive mutation (oma). The mutant mice showed short trunk and short and kinky tail. The skeletal preparations of newborn and prenatal mice showed disorganized vertebrae and numerous vertebral and rib fusions which are thought to be caused by patterning defects at the stage of somitegenesis. Linkage analysis localized the oma locus on the proximal region of mouse chromosome 7 close to Dll3 gene. Dll3 is the gene involved in the Notch signaling pathway and null-mutation of the gene has been reported to cause vertebral deformities. The phenotypic similarity between oma and Dll3 null-mutant mice suggests that the causative gene for the oma mutant is the Dll3 gene. We, therefore, investigated the nucleotide sequence of the Dll3 gene of the oma mouse and found a single nucleotide substitution of G to T which causes missense mutation of glycine to cysteine at codon 409. Since the amino acid substitution is a nonconservative amino acid substitution at the conserved portion of the Dll3 protein, and the substitution is specific to the mutant mice, we concluded that the nucleotide substitution of the Dll3 gene is responsible for the skeletal deformities of the oma mouse.     

Isolation and molecular characterization of bovine Rhesus-like transcripts and chromosome mapping of the relevant locus

The bovine erythrocyte membrane carries Rhesus (Rh)-like proteins. To obtain a bovine nucleotide probe, a cDNA library of foetal liver was constructed and screened with the human RhCE probe. Three clones (245 bp, 1012 bp and 1400 bp) were isolated and sequenced. They share a high degree of similarity (up to 73%) with Rh-like cDNAs of primates characterized so far and all of them were shown to contain a polymorphic microsatellite in their 3' untranslated region. Their sequences support the occurrence of different splicing isoforms transcribed from the same RH-like gene. One of the clones (1400 bp), which has a 134-nucleotide deletion causing a frameshift, is structurally similar to the human Rh4 cDNA isoform. Synteny mapping and genetic linkage analysis located the bovine RH-like locus on chromosome BTA2, on which none of the 10 previously mapped blood group systems are found. In situ hybridization mapped the RH-like locus to BTA2q45. No linkage was detected between the microsatellite and the only unmapped blood group system (locus F). These results strongly suggest that the putative bovine Rh-like polypeptides do not correspond to any previously described bovine blood group. Comparative studies of human and bovine maps clearly show that the human RH locus, which is located on HSA1p34-p36, and its bovine counterpart belong to a linkage group highly conserved between both species.