Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.

Rabbit antibodies to Atlantic cod (Gadus morhua L.) immunoglobulin were affinity purified and used to screen cDNA libraries from spleen and head kidney mRNA. cDNA clones for both the secretory and membrane-bound heavy (H) chain were isolated, the nucleotide and deduced amino acid sequences of which are reported here. Comparisons of the cod secretory H chain amino acid sequence show 24%, 27%, 30% identity to the mu chain of Mus, Xenopus and Ictalurus, respectively. The highest degree of identity was observed in the CH4 domain. The cDNA encoding the transmembrane form shows a novel splicing pattern where the TM1 exon is spliced directly onto the CH3 domain and not to the CH4 domain as in other animal groups. Southern blot analyses with VH and C probes on genomic DNA from cod erythrocytes indicate that there is a unique C gene but several V genes in the cod immunoglobulin H chain locus.     

Isolation and sequence of a cDNA coding for the immunoglobulin mu chain of the sheep.

A sheep cDNA library was screened with a human C mu probe, and the complete nucleotide sequence of a 1923 nt cDNA was determined. It contains sequences corresponding to all the exons (VH, DH, JH, CH1, CH2, CH3 and CH4) characteristic of the immunoglobulin mu heavy chain regions. The deduced amino acid sequence shows a percentage of identical residues in the range 65-45% when compared with the mu chains of various species. The VH region of this clone is clearly related to a group of genes that includes mouse VH36-60 and VHQ52, human VH2, VH4 and VH6 gene families and Xenopus VHII gene families. The constant region shows an unusual repartition of cystein and proline residues at the beginning of the CH2 domain, that may result in a molecule with enhanced stability and reduced flexibility.     

Genomic alterations in a case of alpha heavy chain disease leading to the generation of composite exons from the JH region

Human alpha heavy chain disease (HCD) is characterized by the presence in patient's serum of a short Ig alpha chain devoid of light chains. We analyzed the serum protein, the alpha HCD mRNA and the productive rearranged H chain gene from the leukemic cells of a new case (YAO) of alpha HCD. The abnormal YAO alpha 1 Ig was devoid of VH and CH1 domains and started at the beginning of the hinge region. The alpha HCD mRNA was shorter than normal alpha mRNA and the cDNA prepared from YAO mRNA encoded a leader sequence, an insert of 70 nucleotides and the CH2 and CH3 exons. The origin of the inserted sequence was assessed by cloning and sequence analysis of the alpha 1 productive gene. It started with a leader exon, a leader-VH intron and the first 11 bp of a VH exon. Then the VH region was deleted and replaced by a 19-nucleotide sequence that turned out to correspond to the 3' part of a modified JH5 exon. It was followed by a 221-bp sequence homologous to the JH5-psi JH3 intron and by an inserted sequence of unknown origin. The 3' part of this insertion and the remnant of a JH6 exon delineated a third exon that was followed by a relatively conserved JH6-C alpha intron. These two composite exons were flanked by splicing sites and accounted for the 70-nucleotide insert of the cDNA. The genomic nucleotide sequence also revealed a large deletion in the switch CH1 region which eliminated normal splicing sites and resulted in splicing of the third exon directly to the CH2 exon.     

The human immunoglobulin C mu-C delta locus: complete nucleotide sequence and structural analysis

The entire nucleotide sequence (approximately 20 kbp) spanning the human immunoglobulin IgM (mu) and IgD (delta) heavy chain constant region genes has been determined from DNA of mu-delta producing chronic lymphocytic leukemic B cells. As in the murine IgM + IgD double-producing B cells, no rearrangement has occurred in the C mu-C delta region in the leukemic cells. The C mu locus is highly conserved between mouse and human with the exception of the nucleotide sequence between the C mu 4 and mu M1 exons, which has diverged dramatically. The intergenic sequence between human C mu and C delta is three times larger than the analogous region in the mouse and contains notable features absent from the mouse, including a 443 bp segment that is 96% identical to a 442 bp sequence that occurs just 3' to the heavy chain enhancer, a 366 bp sequence that is directly repeated with 76% homology, and 12 tandem copies of a 35 bp sequence. The human C delta gene contains two additional exons relative to mouse C delta, but shares with the mouse the unique distal location of both secreted and membrane coding segments. Several polymorphisms in the human population have been identified in the intergenic region and in C delta but not in C mu.     

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.     

Molecular and biochemical characterisation of a novel sulphatase gene: Arylsulfatase G (ARSG)

Molecular analysis has provided important insights into the biochemistry and genetics of the sulphatase family of enzymes. Through bioinformatic searches of the EST database, we have identified a novel gene consisting of 11 exons and encoding a 525 aa protein that shares a high degree of sequence similarity with all sulphatases and in particular with arylsulphatases, hence the tentative name Arylsulfatase G (ARSG). The highest homology is shared with Arylsulfatase A, a lysosomal sulphatase which is mutated in metachromatic leukodistrophy, particularly in the amino-terminal region. The 10 amino acids that form the catalytic site are strongly conserved. The murine homologue of Arylsulfatase G gene product shows 87% identity with the human protein. To test the function of this novel gene we transfected the full-length cDNA in Cos7 cells, and detected an Arylsulfatase G precursor protein of 62 kDa. After glycosylation the precursor is maturated in a 70 kDa form, which localises to the endoplasmic reticulum. Northern blot analysis of Arylsulfatase G revealed a ubiquitous expression pattern. We tested the sulphatase activity towards two different artificial substrates 4-methylumbelliferyl (4-MU) sulphate and p-nitrocatechol sulphate, but no arylsulphatase activity was detectable. Further studies are needed to characterise the function of Arylsulfatase G, possibly revealing a novel metabolic pathway.     

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

低密度脂蛋白受体相关蛋白5基因的基因组结构

目的 确定2低密度脂蛋白受体相关蛋白5（low density lipoprotein receptor related protein,5 LRP5）的基因组结构。方法 以LRP5基因的cDNA序列为线索，采用计算机杂交方法首先通过对比分析该基因的cDNA序列和基因组序列，初步确定LRP5基因的基因组结构，按分析得到的基因组结构设计引物，扩增并测定外显子序列和外显子内含子接头序列，确定该基因的基因组结构。结果 LRP5基因的基因组DNA全长为131.6kb，含4有23个外显子和22个内含子；在编码序列中检测到3个单核苷酸多态，即位于第2外显子的A459G，位于第10外显子的C2220T和位于第21外显子的G4416C；LRP5基因含有4个已知的短串联重复序列，即D11S1917，D11S4087，D11S1337和D11S4178，它们分别位于该基因的5'端和第1，4，13内含子内。结论 LRP5基因的基因组结构的确定，为分析该基因突变和功能研究奠定了基础。     

Loss of splice consensus signal is responsible for the removal of the entire C(H)1 domain of the functional camel IGG2A heavy-chain antibodies.

The molecular basis for the absence of the C(H)1 domain in naturally occurring heavy-chain antibodies of the camelids was assessed by determining the entire Camelus dromedarius gamma2a heavy-chain constant gene. The organization of the camel gamma2a constant heavy-chain gene obtained from a liver genomic library appears to be typical of all other mammalian gamma genes sequenced to date. It contains the switch, CH1, hinge, CH2, CH3, M1 and M2 exons. In contrast to the case in mouse and human heavy chain diseases, the camel gamma2a gene shows no major structural defect, and its equivalent CHI exon is intact. However, sequence analysis has revealed that the splicing site, immediately after the CH1 exon, is defective due to point mutations, especially the G(+1) to A(+1) transversion seems to be detrimental. It is concluded that the loss of the splice consensus signal is responsible for the removal of the entire CH1 domain in camel gamma2a heavy-chain immunoglobulins. Additionally, a closer analysis of the hinge exon suggests the possible involvement of transposons in the genetic variation of mammalian Cgamma hinges.     

Properties of B cell stage specific and ubiquitous nuclear factors binding to immunoglobulin heavy chain gene switch regions.

The Ig heavy chain (IgH) constant region (CH) class switch is manifested by DNA deletions which exchange the C mu gene of a functional VDJ-CH rearrangement for a C gamma, C epsilon or C alpha gene. Repetitive sequences (S regions) 5' of each CH gene mediate CH gene switch recombination by an illegitimate mechanism. S mu can be subdivided into S mu 5' (non-repetitive) and S mu 3' (repetitive) components with recombination occurring in either part. Here, we describe the properties of ubiquitous and B cell stage specific S mu binding factors NFS mu-U1 and NFS mu-B1 respectively. U1 only bound to S mu 5' sequences, and B1 to S mu 5', S mu 3' sequences and to other S regions with varying affinities. DMS and OP-Cu footprinting revealed the sequence AAAAAGCATGGCTGA in the U1 site while the B1 S mu 5' site overlapped the 3' end of the U1 binding site and also contained additional 3' flanking S mu repeat motifs (GAGCTGAGATGGGTGGGCT). Binding site competition assays reveal that NFS mu-B1 is either very related or identical to S alpha BP (described by Waters et al., Mol. Cell Biol. 9:5594, 1989) and BSAP (identified by Barberis et al., Genes Devl. 4:849, 1990) which were shown to bind to two sequences upstream of the S alpha repeats and within the promoters of sea urchin histone genes respectively.(ABSTRACT TRUNCATED AT 250 WORDS)