Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.     

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.     

A human immunoglobulinIGHG3 allele (Gmb0, b1, c3, c5, u) with anIGHG4 converted region and three hinge exons

The five humanIGHG genes consist of three constant domain exons plus one of or four hinge exon(s), the quadruplicated hinge region being characteristic of theIGHG3 gene. Besides this structural difference, theIGHG genes are polymorphic, as demonstrated by the restriction fragment length polymorphism and, at the protein level, by the Gm allotypic antigenic determinants. In this paper, we report the sequence of theG3m(b0, b1, c3, c5, u) IGHG3 allele, typical of the Black African populations and of populations with Negroid admixture, found in a homozygous Tunisian designated as LAT. We demonstrate that thisG3 allele contains only three hinge exons instead of four (the probable result of an unequal crossing over) and thatIGHG3 genes with triplicated hinge exons (and therefore encoding shorter γ 3 chains) are present in healthy individuals from different populations. Moreover, we show that the LAT G3m (b0, b1, c3, c5, u) coding sequence results from the conversion, in the CH3 exon, of theG3m (b0, b1, b3, b4, b5, u, v) allele, the most frequentIGHG3 gene in the Negroid populations, by the homologous region of aIGHG4 gene. The structural features of theLAT IGHG3 allele, which are the lack of one hinge exon and its conversion by theIGHG4 gene, demonstrate that both crossing-over and gene conversion events occur in the evolution of the humanIGHG genes. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number X16110.     

Membrane and secretory forms of mouse membrane cofactor protein (CD46) generated from a single gene through alternative splicing

 A cDNA encoding a new secretory form of mouse membrane cofactor protein (MCP, CD46) was identified additionally to the membrane form cDNA. The secretory MCP, predicted from the cDNA sequence, consisted of the conserved four short consensus repeats (SCRs) plus a four amino acid-stretch. Unlike human MCP which comprises many isoforms, mouse MCP cDNA predicted a single isoform of membrane MCP with cytoplasmic tail 1 (CYT1) and serine/threonine-rich domain C (ST^C). To clarify the genomic origin and monomorphic alteration of these cDNAs, we cloned and analyzed a mouse genomic DNA harboring the full coding sequence of MCP from a 129/SV mouse genomic library. The mouse Mcp was a single gene ∼50 kilobases long. Eleven of the 14 coding exons of the human MCP gene and intron-exon boundary sequences were found to be conserved in the mouse gene. The ST^C homologue but not the ST^A or ST^B homologue in the mouse exons was functional: the latter being due to deletions and lack of consensus sequences for splicing. The sequence equivalent to cytoplasmic tail 2 (CYT2) has not been identified in the Mcp genome. Thus, the three exons (ST^A, ST^B, and probably CYT2) responsible for the polymorphism of human MCP by differential splicing were missing in the mouse Mcp gene. Unlike the case in humans, no Mcp-related genes or pseudogenes were observed in the mouse genome. The single mouse Mcp gene was mapped to the R-positive H5 band of mouse Chromosome 1 by FISH. Strikingly, one alternative exon with 73 base pairs (encoding the four new amino acids and a TGA stop codon) was discovered between the SCRIV and the ST^C exons; alternative splicing causes the generation of the secretory form of mouse MCP. These results on mouse MCP, together with the information concerning other mouse SCR proteins, infer that the regulator of complement activation (RCA) gene cluster is genetically diverged between humans and mice. Received: 22 April 1999 / Revised: 21 June 1999     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Colinearity of novel genes in the class II regions of the MHC in mouse and human

To examine the degree of conservation of gene organization in and around the class II regions of the major histocompatibility complexes of mouse and human, we have established the positions of sequences homologous to five human non-class II genes (RING1-5) in mouse, and the positions of sequences homologous to three mouse non-class II genes (KE3-5) in human. The resulting comparative map reveals that the organization of genes in the entire proximal region of the MHCs of mouse and human is remarkably conserved, apart from the H-2K gene pair in mouse, which can be accounted for by a 60 kilobase (kb) insertion. The characterization of the novel human gene RING5 is also presented. This gene, which is widely expressed, maps 85 kb proximal to the DPB2 gene. Partial nucleotide sequencing of a RING5 cDNA clone reveals that it is the human homolog of the mouse KE4 gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M58660.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.     

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrats considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dCdA)₁₅, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M33306 and M33307. Address correspondence and offprint requests to: M. R. Brandon.     

Human fibroblast growth factor receptor 3 gene (FGFR3): genomic sequence and primer set information for gene analysis

We have determined the nucleotide sequence of the human fibroblast growth factor receptor 3 (FGFR3) gene, including 800 bp of the 5′-flanking region and compared the sequence with the previously published murine Fgfr3 gene. The organization of the gene is highly conserved between man and mouse. We used the intron sequences to design a set of primers that allow amplification of the 17 exons (2–18) that encode the complete open reading frame. Using these primers the FGFR3 gene can be amplified at the genomic level, which significantly facilitates mutational screening. Received: 27 December 1996 / Accepted: 6 March 1997     

The structure of the mouse immunoglobulin in gamma 3 membrane gene segment

The genomic region containing the mouse immunoglobulin gamma 3 heavy chain membrane (M) exons has been located and sequenced. The exon structure is highly similar to that of the other mouse gamma chains, with strong sequence conservation in the coding regions and the intron 5' to the M1 exon. The intron between M1 and M2 shows moderate sequence homology but very strong conservation of size. RNA blots suggest that gamma 3 membrane exon usage is similar to that seen in other immunoglobulin membrane heavy chain mRNAs. The transmembrane region contains the invariant residues which have been noted in all other heavy chain sequences and which were previously proposed to be interactive in a two-chain model for insertion through the lymphocyte membrane. Conserved residues with similar spacing have been seen in class II histocompatibility antigens, which are also two-chain transmembrane molecules, but not in class I antigens, which span cell membranes with a single chain.     

Polymorphism and conservation of the genes encoding Qa1 molecules

To evaluate the polymorphism and conservation of the major histocompatibility complex class Ib molecule Qa1 in wild mouse populations, we determined the nucleotide sequence of exons 1–3 of Qa1 of eight mouse haplotypes derived from wild mice, including Mus musculus domesticus, M. m. castaneus, M. m. bactrianus, and M. spretus, as well as two t haplotypes. Our data identify eight new alleles of Qa1. Taken together with previously published data on Qa1 among the common laboratory inbred strains, and in agreement with cytotoxic T-lymphocyte, serological, and biochemical data, these results further confirm the existence of two families of Qa1 molecules, Qa1^a-like and Qa1^b-like, and illuminate the extreme conservation of the peptide-binding region of these molecules, even across species.The wild mouse Qa1 nucleotide sequences are available from GenBank at accession numbers AF100695–703     

Characterization of the Gene Encoding Human Sarcolipin (SLN), a Proteolipid Associated with SERCA1: Absence of Structural Mutations in Five Patients with Brody Disease

Sarcolipin (SLN) is a low-molecular-weight protein that copurifies with the fast-twitch skeletal muscle sarcoplasmic reticulum Ca²⁺ATPase (SERCA1). Genomic DNA and cDNA encoding human sarcolipin (SLN) were isolated and characterized and theSLNgene was mapped to chromosome 11q22–q23. Human, rabbit, and mouse cDNAs encode a protein of 31 amino acids. Homology of SLN with phospholamban (PLN) suggests that the first 7 hydrophilic amino acids are cytoplasmic, the next 19 hydrophobic amino acids form a single transmembrane helix, and the last 5 hydrophilic amino acids are lumenal. The cytoplasmic and transmembrane sequences are not well conserved among the three species, but the lumenal sequence is highly conserved. Like SERCA1, SLN is highly expressed in rabbit fast-twitch skeletal muscle, but it is expressed to a lower extent in slow-twitch muscle and to an even lower extent in cardiac muscle, where SERCA2a and PLN are highly expressed. It is expressed in only trace amounts in pancreas and prostate.SLNandPLNgenes resemble each other in having two small exons, with their entire coding sequences lying in exon 2 and a large intron separating the two segments. Brody disease is an inherited disorder of skeletal muscle function, characterized by exercise-induced impairment of muscle relaxation. Mutations in theATP2A1gene encoding SERCA1 have been associated with the autosomal recessive inheritance of Brody disease in three families, but not with autosomal dominant inheritance of the disease. A search for mutations in theSLNgene in five Brody families, four of which were not linked toATP2A1,did not reveal any alterations in coding, splice junction or promoter sequences. The homozygous deletion of C438 in the coding sequence ofATP2A1in Brody disease family 3, leading to a frameshift and truncation following Pro₁₄₇in SERCA1, is the fourthATP2A1mutation to be associated with autosomal recessive Brody disease.