The genomic locus of the human hemopoietic-specific cell protein tyrosine kinase (PTK)-encoding gene (HCK) confirms conservation of exon-intron structure among human PTKs of the src family.

Protein tyrosine kinases (PTKs) are implicated in the control of cell growth by virtue of their frequent appearance as products of retroviral oncogenes, as intracellular signal transducers, and as growth factor receptors or components thereof. The knowledge of the structure and sequence of family genes encoding PTKs is still limited. To date, the complete genomic structure of human src family members is only available for the C-FGR gene (encoding p55 Fgr, PTK). Sequence analysis and characterization of the intron/exon organization of the human HCK gene, encoding a hemopoietic-specific cell PTK of the src-related family, revealed a length of over 16 kb for the seven 3'-exons. All intron/exon splice junctions agree with the GT/AG rule. In each case where a boundary occurs at a Gly codon, GGG or GGA, the triplet is split between the first and second nucleotide (nt). A total of eight complete and one partial Alu repeats were identified within the introns. The nt sequence of the genomic clones resolves existing discrepancies among two published sequences of HCK cDNAs. Human HCK, C-SRC (encoding p60 Src PTK), C-FGR and LCK (encoding p56 Lck, PTK) genes thus share very similar exon/intron structures for the conserved exons. These results provide additional evidence that the different PTKs of the src-like family most likely arose by duplication of an ancestral src-like gene.     

Structure of human immunoglobulin gamma genes: implications for evolution of a gene family

We have cloned five human immunoglobulin gamma genes from a fetal liver gene library. Four of them encode the known human immunoglobulin gamma chains gamma 1, gamma 2, gamma 3 and gamma 4. A fifth gamma gene seems to be a pseudogene. Nucleotide sequence determination demonstrates that the gamma 3 gene contains four separate hinge exons. Comparison of these hinge exons with those of the other gamma genes indicates that the first hinge exon is homologous to that of the pseudogene, and that the other three hinge exons are homologous to that of the gamma 1 gene, suggesting that the gamma 3 gene ancestor is a hybrid gene created by unequal crossing-over between the ancestral gamma 1 and psi gamma genes. Amplification of the gamma 1-type hinge exon probably followed to complete the gamma 3 gene. This hypothesis inevitably postulates the gene order 5'-gamma 1-gamma 3-psi gamma-3'. Cloning of overlapping chromosomal segments demonstrates that the gamma 2 gene is located 19 kb 5' to the gamma 4 gene. These analyses indicate that the human gamma-gene family has evolved by several types of DNA rearrangemet, including duplication of a complete gene; duplication of a hinge exon; and reassortment of exons by unequal cross-over between two adjacent genes.     

Genomic Structure and Chromosomal Localization of Mouse Cyclin G1 Gene

Mouse genomic DNA harboring the full coding sequence of cyclin G1 was cloned and analyzed. The locations of five coding exons and the intron–exon boundary sequences were found to be conserved between the mouse and the human genes. Two putative binding sites for thep53tumor suppressor gene product were found around the first exon: one was located in the 5′ regulatory region, and the other was in the first intron. The mouse cyclin G1 gene was mapped to bands A5 to B1 of chromosomes 11 (11A5–B1) by FISH using genomic DNA clone as a biotinylated probe. The location of mouse cyclin G1 is syntenic to that of its human homologue, which we previously mapped to 5q32–q34 of chromosome 5. An additional faint signal was detected on chromosome 4 (4B1–C2), probably indicating the presence of a cyclin G1-related gene or pseudogene in the mouse genome.     

Structure of the mouse serum amyloid P component gene

A genomic DNA clone corresponding to the mouse serum amyloid P component (SAP) has been isolated and characterized for the first time. The numbers of exons, the relative sites of intron/exon junctions, and the size of the coding region for mature SAP protein are all in complete agreement with those of the human SAP gene. In the 5'-flanking region of the mouse SAP gene, there is a small DNA segment (43-base pairs) which is highly homologous with the corresponding region of the human SAP gene. However, most parts of the 5'-flanking regions are not conserved between the mouse and human SAP genes, and several phorbol ester-responsive element-like sequences are present only in the mouse SAP gene.     

Genomic organisation of the spinocerebellar ataxia type 7 (SCA7) gene responsible for autosomal dominant cerebellar ataxia with retinal degeneration

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) is a progressive neurodegenerative disorder caused by a CAG expansion in the spinocerebellar ataxia 7 (SCA7) gene. Here, we describe the genomic organisation of the human SCA7 gene. The exon-intron boundaries were identified by sequencing plasmid subclones of a P1 artificial chromosome (PAC) clone containing the entire SCA7 gene. We found 13 exons, ranging in size from 69 to 979 bp, with all exon-intron boundaries following the GT-AG rule. The ATG initiation codon at position 554 of the cDNA occurs in exon 3 at position 12 and the coding region extends to the first five codons of exon 13, with the CAG repeat being located in exon 3 starting at codon 30. The intron sizes were determined by long-distance polymerase chain reaction with primers from neighbouring exons and by restriction mapping of the SCA7 PAC clone. The introns varied in size from 233 bp to about 40 kb, resulting in an overall size estimate for the SCA7 gene of 140 kb. Sequence analysis of intron 7 (491 bp) revealed a polymorphic GT/AC repeat, a useful intragenic marker for SCA7 in segregation studies.     

Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana

Gene BoGSL-PRO is associated with presence of 3-carbon side-chain glucosinolates (GSL). This gene is a member of the methylthioalkylmalate synthase (MAM) gene family. A BAC clone of Brassica oleracea, B21F5, containing this gene, was sequenced, annotated and compared to its corresponding region in Arabidopsis thaliana. Twelve protein-coding genes and 10 transposable elements were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no transposable elements. Analysis of MAM gene family in both species, which also include genes controlling 4-carbon side-chain GSL, separated the genes in two groups based on exon numbers and function. Phylogenetic analysis of the amino acid sequences encoded by these genes suggest that these two groups were produced by a duplication that must have occurred before the divergence of the Rosid and Asterid lineages of angiosperms. Comparison with putative orthologs from several prokaryotes further suggest that the members of the gene family with 10 exons, which encode proteins involved in 4-carbon side-chain GSL biosynthesis, were derived via truncation of the 3′ end from ancestral genes more similar in length to those with 12 exons, which encode proteins involved in 3-carbon side-chain GSL biosynthesis. Lower gene density in B. oleracea compared to A. thaliana is due in part to presence of transposable elements (TE) mostly in inter-genic regions.     

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.