Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Orientation and molecular map position of the complement genes in the mouse MHC.

Over the past few years six gene clusters have been isolated from the major histocompatibility complex (MHC) of the BALB/c mouse encompassing a total of 1600 kb of DNA and 48 genes. The molecular distances between these gene clusters and the orientation of four of the six clusters on chromosome 17 is not known. Here we use pulse-field gradient gels and Southern blot hybridization to establish large-scale genomic restriction maps covering several hundreds of kb surrounding the three gene clusters located in the K, I, S, and D regions of the MHC. Comparison of the maps orients the complement gene clusters in the S region with the 21-OHB gene pointing towards the K end and the C2 gene pointing towards the D end of the MHC. The distances between the E alpha and 21-OHB genes is 430 kb and between the C2 and TNF-alpha genes at least 420 kb.     

Immunoglobulin genomics in the guinea pig (Cavia porcellus)

In science, the guinea pig is known as one of the gold standards for modeling human disease. It is especially important as a molecular and cellular biology model for studying the human immune system, as its immunological genes are more similar to human genes than are those of mice. The utility of the guinea pig as a model organism can be further enhanced by further characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the guinea pig immunoglobulin (Ig) heavy and light chain genes. The guinea pig IgH locus is located in genomic scaffolds 54 and 75, and spans approximately 6,480 kb. 507 V(H) segments (94 potentially functional genes and 413 pseudogenes), 41 D(H) segments, six J(H) segments, four constant region genes (μ, γ, ε, and α), and one reverse δ remnant fragment were identified within the two scaffolds. Many V(H) pseudogenes were found within the guinea pig, and likely constituted a potential donor pool for gene conversion during evolution. The Igκ locus mapped to a 4,029 kb region of scaffold 37 and 24 is composed of 349 V(κ) (111 potentially functional genes and 238 pseudogenes), three J(κ) and one C(κ) genes. The Igλ locus spans 1,642 kb in scaffold 4 and consists of 142 V(λ) (58 potentially functional genes and 84 pseudogenes) and 11 J(λ) -C(λ) clusters. Phylogenetic analysis suggested the guinea pig's large germline V(H) gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves.     

Structure and chromosomal localization of the gene for the oligodendrocyte-myelin glycoprotein

Utilizing a cDNA clone encoding the oligodendrocyte-myelin glycoprotein (OMgp) to screen a human genomic DNA library, we have obtained a clone that contains the OMgp gene. The genomic clone was restriction mapped and the OMgp gene and its 5' and 3' flanking regions were sequenced. A single intron is found in the 5' untranslated region of the gene, while the coding region is uninterrupted by an intron. This placement of a single intron in the OMgp gene is identical to that of the gene for the alpha-chain of platelet glycoprotein Ib, which, along with OMgp, belongs to a family of proteins sharing two distinct structural domains: an NH2-terminal cysteine-rich domain and an adjacent domain of tandem leucine-rich repeats. Hence, it is possible that this family of proteins is not only related in terms of primary structure, but also through similar gene structure. Sequence comparison of the 5' and 3' flanking regions did not reveal striking similarities to other DNA sequences, and no obvious promoter elements were noted. By hybridization of the genomic clone to metaphase cells, we have localized the human OMgp gene to chromosome 17 bands q11-12, a region to which the neurofibromatosis type 1 gene has been previously mapped.     

Cloning and characterization of the 5' end (exon 1) of the gene encoding human factor X

Human blood-coagulation factor X (hBCFX) is a serine protease zymogen which participates in the middle phase of blood coagulation. Recently, we and others have reported the cDNA sequences. At present, partial hBCFX gene structure is available. In this paper, we report the isolation of two genomic clones, the X-emb lambda phage clone encoding exons 1 and 2 of the hBCFX gene, and the X-cos cosmid clone encoding exons 2-8. The restriction map of the hBCFX region spans 55 kb. The gene itself was found to be 27 kb long. The sequence of the 5' region of the hBCFX gene has been determined and reveals an ATTTG pentanucleotide, which also occurs in a similar location in the genes encoding factors VII, IX, protein CC and C prothrombin, suggesting that this motif might be of importance in the regulation of these genes.     

Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6

On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21-q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5' UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K(a)/K(s) ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs.     

Isolation of four HSP70 genes in the pig and localization on Chromosomes 7 and 14

For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5 section, the central section, and the 3 section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4–2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.     

Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization

Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons. Only the latter half of the second SCR was present on the clone, and it was encoded by a single exon, demonstrating that murine C4BP has a split SCR at the genomic level. Structural mapping of this portion of the gene demonstrates that the region extending from the second half of the second SCR through the nonrepeat and untranslated region spans approximately 12 kb; however, genomic Southern blot analysis suggests that the gene is between 20 and 30 kb in length. Analysis of the 3' genomic sequence demonstrates that this region of the gene has homology with SV-40 late (class II) RNA sequences. These sequences may play a role in 3' cleavage of the precursor RNA prior to polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat copper/zinc superoxide dismutase gene: isolation, characterization, and species comparison.

A 13 kb rat Cu/ZnSOD genomic clone has been purified from a rat liver genomic library and completely characterized by restriction mapping, detailed sequencing and Southern blot analysis. This gene spans approximately 6 kb and contains five exons and four introns. Comparison of rat, mouse, and human Cu/ZnSOD genes reveals a high conservation in genomic organization and exon-intron junctions, including an unusual 5'GC donor sequence at the first intron. The gene contains a TATA box as well as an inverted CCAAT box, a feature common to both the mouse and human genes. Furthermore, several repeats were identified in the 5' promoter region of this gene, and these regulatory elements are also strikingly conserved in these three species.     

Physical and cDNA mapping in the DBH region of human chromosome 9q34

Chromosome 9q34 has been extensively studied and mapped due to the presence of known disease genes, principally tuberous sclerosis 1 (TSC1), in this region. During the course of our mapping of this region we constructed a 555-kb contig beginning approximately 50 kb proximal to the dopamine-beta-hydroxylase (DBH) gene and extending, with one small deletion, distal to the D9S114 marker. The contig consists of 11 P1 clones, four PAC clones, one BAC clone and six cosmid clones and contains 27 new nonpolymorphic STSs. We have found the region to be unstable in P1, PAC and BAC cloning vehicles and have identified several deleted genomic clones. In addition, we have isolated and mapped the 3' portions of three putative genes located within or immediately distal to the DBH gene, including one large gene that runs on the opposite strand to DBH and utilizes portions of two DBH exons. The genomic clones of the contig, cDNAs and new STSs will be useful reagents for the further study and mapping of this region.