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.     

Murine enamelin: cDNA and derived protein sequences

Enamelin is the largest enamel protein. Recently we reported the characterization of a cDNA clone encoding porcine enamelin. The secreted protein has 1104 amino acids--over 6 times the length of amelogenin (173 amino acids) and almost 3 times the lengths of sheathlin (395 amino acids) and tuftelin (389 amino acids). Immunohistochemistry has shown that uncleaved porcine enamelin concentrates at the growing tips of the enamel crystallites while its cleavage products localize to rod and interrod enamel. Here we report the isolation and characterization of cDNA encoding murine amelogenin and demonstrate the tooth specificity of porcine enamelin. The murine clone is 4154 nucleotides in length and encodes a protein of 1274 amino acids. In the absence of post-translational modifications murine enamelin has an isotope averaged molecular mass of 137 kDa and an isoelectric point of 9.4. Multiple tissue Northern blot analyses detect porcine enamelin mRNA in developing teeth but not in liver, heart, brain, spleen, skeletal muscle and lung. Mouse and porcine enamelin share 61% amino acid identity and 75% DNA sequence identity. Mouse enamelin has 14 tandemly arranged copies of an 11 amino acid segment that is found only once in porcine enamelin.     

Molecular cloning of the cDNA encoding the constant region of the immunoglobulin A heavy chain (Cα) from a marsupial: Trichosurus vulpecula (common brushtail possum)

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (Cα) was isolated by screening a mesenteric lymph node cDNA library with a porcine Cα exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian Cα sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum Cα sequence had a nucleotide sequence identity of 57.7% with porcine Cα, 51% with mouse Cα, 46.7% with dog Cα and 45.9% with human Cα2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.     

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene

Summary The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.GenBank Accession No. U24432.     

Nucleotide sequence of a cDNA encoding the constant region of a rabbit immunoglobulin light chain of the lambda type

A cDNA library has been constructed in the plasmid pBR322 using as template 12 S poly(A)-RNA isolated from spleen cells of a hyperimmunized Basilea rabbit. One of these cDNA-containing clones was used to determine the nucleotide sequence coding for the lambda light chain constant (C) region. The deduced amino acid sequence of this cDNA was found in good agreement with a Basilea rabbit C lambda region amino acid sequence previously determined. The nucleotide sequence of the rabbit C lambda-coding region was compared with man, mouse and chicken C lambda sequences and showed 78%, 72% and 66% homology, respectively. Southern blot hybridization analyses of liver DNA from various rabbits were carried out. The comparison of the restriction patterns suggests that a few C lambda-related genes occur in the rabbit genome. In addition, discrete differences in the restriction patterns may exist between rabbits of different genetic backgrounds.     

Sequence Analysis of the Gene Encoding VP4 of a Bovine Group C Rotavirus: Molecular Evidence for a New P Genotype

Nucleotide sequence of the bovine group C rotavirus Shintoku strain gene 3 was determined. Segment 3 is 2253 nucleotides (nt) in length and contains a long open reading frame (ORF) beginning at nt 22 and terminating at nt 2223. This ORF encodes a polypeptide of 733 amino acids with a predicted molecular mass of 83 kDa. The deduced gene 3 amino acid sequence shares 79% and 73% identities with VP4 of the porcine Cowden and human Bristol strains, respectively. Lack of high amino acid sequence homology in VP4 of bovine, porcine, and human group C rotaviruses indicates that the Shintoku strain represents a new P genotype.     

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belém VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.     

Molecular cloning of the cDNA coding for properdin, a positive regulator of the alternative pathway of human complement

Northern blot analysis indicated that the mRNA for human properdin is approximately 1.5 kb long and that its level in U-937 cells is increased by pretreating the cells with phorbol 12-myristate 13-acetate (PMA). Using a human genomic probe clones coding for human properdin were isolated from a lambda gt10 cDNA library derived from PMA-treated U-937 cells. The sequence of the 1474-bp cDNA insert of the longest clone revealed an open reading fram of 1326 bp coding for the entire 442 amino acids of the mature form of human properdin and 67 bp coding for 22 amino acids of typical, but incomplete leader sequence. Polymerase chain reaction "RACE" experiments identified the start site ATG and revealed the complete, 27-amino acid-long, leader peptide sequence. Within the 81-bp 3' non-translated extension a polyadenylation signal was identified 41 bp downstream from the stop codon, TAA, and 12 bp upstream of a 19 nucleotide long poly(A) tail. The amino acid sequence of human properdin is clearly divided into three distinct regions: a 49 residue-long N-terminal region, a 32 residue-long C-terminal region and a middle region, covering residues 50 to 411, composed of six tandemly repeated thrombospondin repeat (TSR) motifs of the type first described in the adhesive glycoprotein thrombospondin and also known to be present in the C6, C7, C8 alpha, C8 beta and C9 terminal components of complement. Human and mouse properdin sequences show a high (approximately 76%) degree of identity with almost complete conservation of the relatively large number of Cys (44) and Trp (20) residues.     

Shark complement: an assessment

Summary: The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA done encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish ( Triakis scyllia ) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C5n and C9n, and is activated by immune complexes in the presence of Ca⁺⁺ and Mg⁺⁺ ions. The ACP is antibody independent, requiring Mg⁺⁺ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C1 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q like rather than the mannan-binding protein. N-terminal amino acid sequences of the α and β chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4γ chain, shows little similarity to human C4γ chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg Induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and al-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.     

中国大陆庚型肝炎病毒NS区部分基因序列分析及变异 …

目的 研究中国庚型肝炎患者分离的病毒基因的特异性及变异规律。方法 应用逆转录－巢式聚合酶链反应（ＰＣＲ）技术从１６例庚型肝炎病毒（ＨＧＶ）感染住院病人血清中扩增了ＨＧＶＮＳ５区部分基因片段。经纯化后采用双脱氧链末端终止法进行序列分析。     

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.     

Characterization of the gene for the membrane and secretory form of the IgM heavy-chain constant region gene (C mu) of the cow (Bos taurus).

Our present understanding of the evolution of immunoglobulins is derived from a few vertebrate species. In order to obtain additional information on the development of the humoral immune system, we cloned and determined the nucleotide sequence of the bovine cDNA and genomic IgM heavy-chain constant region gene (C mu). The gene contains four constant region domain-encoding exons (CH1 to CH4) and two exons encoding the transmembrane domain (TM1, TM2), expressed in the membrane-bound receptor form of the IgM. The sequence of a cDNA clone encoding the 3' portion of the membrane form of the mu-chain revealed that the TM1 exon is spliced to the CH4 exon, as occurs in other mammals. Comparison of deduced amino acid sequence data from different vertebrates revealed a high similarity to sheep C mu (88%) and a lower degree of similarity to pig (62%), rat (62%), rabbit (58%) human (56%), hamster (55%), mouse (54%), chicken (28%) and horned shark (22%) C mu.     

A pre-translational defect in a case of human mu heavy chain disease

A patient (BW) was studied with Mu heavy chain disease (mu HCD) in whom a leukemic B-cell clone secreted a shortened monoclonal mu chain without associated light chain. The cells did, however, produce a normal-sized kappa light chain that was detected as urinary Bence-Jones protein. The cytoplasmic and secreted monomeric mu chain had an approximate mol. wt of 58,000. Radiochemical sequence analysis of the biosynthetically labelled mu chain revealed a protein that lacked the entire variable region. The sequence initiated at amino acid position 5 within the first constant region domain (CH1) of C mu. The primary in vitro translation product, the cytoplasmic and secreted proteins were all similarly truncated, thereby excluding extensive postsynthetic degradation. The mu RNA, that directed the synthesis of the truncated mu protein, was about 350 bp smaller than the normal mu RNA. Furthermore, by primer extension analysis it was possible to localize this deletion in the mu RNA to a region 5' of CH1. Thus, a defect at the level of Ig gene structure/assembly that deletes coding information or results in aberrant RNA processing must be responsible for the truncated mu HCD protein BW.