Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.     

Molecular characterization of the Japanese flounder, Paralichthys olivaceus, CD3epsilon and evolution of the CD3 cluster

A second CD3 gene, i.e. CD3epsilon, has been cloned and sequenced in Japanese flounder. The full length cDNA is 1006 bp and encodes 164 amino acids. When compared with other known CD3epsilon peptide sequences, the most conserved region of the Japanese flounder CD3epsilon chain peptide is the cytoplasmic domain and the least conserved is the extracellular domain. A phylogenetic analysis based on the deduced amino acid sequence grouped the two Japanese flounder CD3 sequences with CD3epsilon and CD3gamma(delta, respectively. The Japanese flounder CD3epsilon gene has Lyf-1, GATAs, Oct-1, CEBPs, AP-1, and NF-AT but lacks TATA and CCAAT elements in the 5' flanking region. The Japanese flounder CD3 cluster (consisting of CD3epsilon and CD3gamma(delta) spans only 10.4 kb. The two genes are oppositely transcribed only 3.8 kb apart. Both Japanese flounder CD3 genes have five exons. The two Japanese flounder CD3 genes were predominantly expressed in PBLs, kidney, spleen, and gills. A polyclonal rabbit antiserum that reacts with the CD3 marker on human T cells also reacted with Japanese flounder CD3epsilon. The epitope highly conserved between mammalian and non-mammalian CD3epsilons, this antibody bound to a single 15 kDa peptide.     

Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes

The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.     

用标记磁微粒技术克隆牛CD14基因

为获得牛ＣＤ１４ 的全长编码基因，以探讨牛ＣＤ１４ 基因结构与功能的关系，进一步阐明其作用机理，我们用抗牛ＣＤ１４ 的单克隆抗体（ｍＡｂ）ＣＣＧ３３ 标记的磁微粒作为探针，从转染有牛肺泡巨噬细胞ｃＤＮＡ文库的ＣＯＳ７细胞中进行筛选。并对获得的牛ＣＤ１４ ｃＤＮＡ进行了克隆和序列分析。结果表明，牛ＣＤ１４ 基因全长为１１２２ ｂｐ，共编码３７３ 个氨基酸，其中含有信号肽序列和３ 个可识别的Ｎ连接糖基化位点，但缺少穿膜结构域及细胞内区。证实牛ＣＤ１４ 以糖基磷脂酰肌醇（ＧＰＩ） 锚定在靶细胞膜上；其氨基酸序列与人和小鼠ＣＤ１４ 的同源性分别为７３－５ ％ 和６１－４ ％ ，三者共有的保守序列约为５５％ 。     

cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide; all of the 228 amino acids of the mature protein; and 140 nucleotides of the 3' untranslated region, including a poly A additional signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the human B chain (Reid, K.B.M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.     

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.     

cDNA clones coding for the complete murine B chain of complement C1q: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit C1q from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of C1q. By comparing the nucleotide sequence of the mouse B chain of C1q with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.     

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.     

Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein alpha3 (cpalpha3)

We report here the molecular cloning and characterization of a novel human actin capping protein alpha3 (cpalpha3) cDNA, an orthologue of the mouse male germ cell-specific cpalpha3, and the organization of the human cpalpha3 genomic structure. The entire coding region of the human cpalpha3 cDNA showed 82.1% similarity with the mouse cpalpha3. The predicted amino acid sequence was 91.3% identical to the mouse protein and the actin-binding motif in the C-terminal region is highly conserved among species. The mRNA of the human cpalpha3 gene was found to be exclusively expressed in the testis. Western blot analysis detected a 33 kDa protein in human testis and sperm. Immunohistochemistry showed that the main localization of human CPalpha3 protein was in the neck region of ejaculated sperm, with moderate and faint signals also detected in the tail and postacrosome region respectively. Furthermore the localization of CPalpha3 coincided with the species-specific distribution of actin in human sperm. The human cpalpha3 gene was mapped to chromosome 12p12 by computer database cloning of human genomic DNA and was proven to be intronless. CPalpha3 may play a physiologically important role in sperm architecture as well as in fertility of the human male.     

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.     

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.     

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.     

Search for a Caenorhabditis elegans FMR1 homologue: identification of a new putative RNA-binding protein (PRP-1) that hybridizes to the mouse FMR1 double K homology domain.

A mixed stage lambdagt10 Caenorhabditis elegans cDNA library was screened with a probe derived by polymerase chain reaction from the double K homology (KH) domain of mouse FMR1 cDNA, a region that is highly conserved in the human, mouse, chicken, and frog FMR1 proteins. Four positively hybridizing cDNAs were cloned and characterized by sequencing. The overlapping sequences map to cosmid R119 from C. elegans linkage group (chromosome) I, and encode a novel proline-, polyglutamine-, and RGG box-rich putative RNA-binding protein. While the cDNA has two regions with similarity to the mouse double KH domain probe at the nucleotide level, there is no significant similarity of the amino acid sequence with human FMR1, FXR1 or FXR2, nor with KH amino acid motifs. The R119 protein, therefore, does not represent an FMR1 homologue.     

Nucleotide sequence of cDNA and derived amino acid sequence of rabbit complement component C3 alpha-chain

The nucleotide sequence coding for 726 amino acid residues of the alpha-chain of rabbit C3 was determined from a cDNA clone. Subfragments of the cDNA produced by restriction endonucleases were inserted into the bacteriophage M13 and sequenced using the dideoxynucleotide technique. The derived amino acid sequence was compared with those of human and mouse C3, which have been previously reported [by De Brujn, M.H.L. and Fey, G.H. (1985) Proc. Natl. Acad. sci. USA 82, 708, and Westel, R.A. et al. (1984) J. Biol. chem. 259, 13857, respectively]. There was 79% or 78% homology in amino acid sequence between rabbit and human or mouse C3, respectively. All of the cysteinyl residues were conserved among the three molecules, and the sequence around the thioester site was also highly conserved. Several regions having low homology were found: one of them was the small fragment released by factor I cleavage.