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.     

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.     

Nucleotide sequence and structure of integrated bovine leukemia virus long terminal repeats

Bovine leukemia virus (BLV) proviruses, harbored by the productively infected fetal lamb kidney (FLK-BLV) cell line, were cloned in bacteriophage lambda L47. The nucleotide sequence of the proviral long terminal repeats (LTR) with flanking cell and virus DNA have been determined. The BLV LTR is 531 bp in length and is bounded by the dinucleotides 5'-TG...CA-3', which are part of a 3-bp inverted repeat. The integrated provirus is flanked by 6-bp direct repeats of cellular DNA. A tRNApro primer binding site is present starting 2 bp downstream of the 5' LTR. In addition to sequencing integrated proviral DNA clones, the nucleotide sequence of a cDNA clone, representing the 3' end of genomic viral RNA, was determined; thus revealing the RNA polyadenylation site and R:U5 boundary within the LTR. Unlike most other retroviruses, a consensus polyadenylation signal, "AATAAA," is not located proximal to the BLV polyadenylation site. The RNA initiation site, defining the U3:R boundary, was located in the BLV LTR by S1 nuclease mapping. This site is approximately 25 bp downstream of an A + T-rich region which probably encompasses a Goldberg-Hogness ("TATAA") box and about 90 bp downstream of a potential "CCAAT" box. The BLV LTR possesses a U3 region of 204 bp, an unusually long R region of 241 bp, and a U5 region of 86 bp.     

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid sequencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was identified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N-terminal extracellular protein with two cysteine-rich domains, three N-linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (omega) site. Database searches revealed homology to Ly-6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly-6 snake toxin superfamily.     

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.     

Specific inhibition of class II MHC gene expression by anti-sense RNA

We have established an anti-sense RNA system which is capable of regulating expression of the class II (Ia) molecule coded for by the major histocompatibility complex in cultured mouse cells. Various areas of the I-A beta chain gene were subcloned in an anti-sense orientation to the 3' of the dihydrofolate reductase (DHFR) cDNA under the control of the human metallothionein IIa gene promoter. These anti-sense DNA constructs were transfected into M12.4 cells, a BALB/c B lymphoma cell line which expresses both I-A and I-E molecules on the cell surface. I-A expression of selected clones transfected with anti-sense DNA encompassing the 5' untranslated region (UT) (100 or 310 bp) including the translation start site or the poly(A) addition signalling sequence in the 3' UT (250 bp) of the I-A beta chain gene were specifically reduced to less than 5% of the control M12.4 cell surface I-A expression. These clones had normal levels of I-E expression. However, transfection of the anti-sense DNA to the beta 1 domain (510 bp) including the splicing donor and acceptor sequences did not affect the expression of I-A molecules. The same antisense DNA constructs (100 bp of the 5' UT or 250 bp of the 3' UT) without the DHFR cDNA (710 bp) did not down-regulate the expression of I-A molecules, indicating that either the physical length of the anti-sense RNA or specific DHFR cDNA sequences are also important.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slc19a2: cloning and characterization of the murine thiamin transporter cDNA and genomic sequence, the orthologue of the human TRMA gene

Recently, our group and others cloned the TRMA disease gene, SLC19A2, which encodes a thiamin transporter. Here, we report the cloning and characterization of the full-length cDNA and genomic sequences of mouse Slc19a2. The Slc19a2 cDNA contained a 1494-bp open-reading frame, and had 5'- and 3'-untranslated regions of 189 and 1857 bp, respectively. A putative GC-rich, TATA-less promoter was identified in genomic sequence directly upstream of the identified 5' end. The Slc19a2 gene spanned 16.3 kb and was organized into six exons, a gene structure conserved with the human orthologue. The predicted Slc19a2 protein, like SLC19A2, was predicted to have 12 transmembrane domains and shared a number of other conserved sequence motifs with the human orthologue, including one potential N-glycosylation site (N(63)) and several potential phosphorylation sites. Comparison of the Slc19a2 amino acid sequence with those of the other known SLC19A solute carriers highlighted interesting patterns of conservation and divergence in various domains, allowing insight into potential structure-function relationships. The identification of the mouse Slc19a2 cDNA and genomic sequences will facilitate the generation of an animal model of TRMA, permitting future studies of disease pathogenesis.     

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 Coccidioides immitis antigen 2 cDNA.

Previous experiments have provided evidence that Coccidioides immitis antigen 2 (Ag2) is a major T-cell-reactive component of mycelia and spherule cell walls. Here we report the identification and cloning of the cDNA that encodes Ag2 from a lambda ZAP cDNA expression library constructed from spherule-derived RNA. DNA sequence analysis established that the 1,255-bp clone contains a 174-bp 5' untranslated region, a 582-bp open reading frame which encodes for a protein consisting of 194 amino acids, and a 375-bp 3' untranslated region, including a poly(A) tail. The recombinant Ag2 protein has a predicted molecular mass of 19.5 kDa and contains an 18-amino-acid N terminus which has been tentatively identified as a signal peptide. The Ag2 cDNA was ligated into the pGEX-4T-3 vector and expressed in Escherichia coli TG-1 cells as a glutathione S-transferase fusion protein. The recombinant fusion protein showed reactivity with sera from patients with coccidioidomycosis and elicited delayed-type footpad hypersensitivity responses in Coccidioides-immune mice. These results suggest that the Ag2 cDNA can be used for the large-scale production of this immunologically important protein.     

中国人群IL-1R1基因变异及其对跨膜区功能影响的预测

目的　检测中国人白细胞介素 1Ⅰ型受体基因 (interleukin 1receptortypeⅠ ,IL 1)调控区和编码区的单核苷酸多态性 (singlenucleotidepolymorphisms ,SNPs) ,并初步探讨其可能对中国人IL 1R1的功能影响。方法　采用直接测序的方法检测基因的 5′区、编码区、部分内含子区和 3′区 ,以确定中国人群中IL 1R1基因SNP的位置和类型 ,用生物信息学方法对编码区SNP的功能进行了预测。结果　在 964 3bp的测序长度中 ,共发现了 16个SNP ,包括 5′区 4个 ,内含子区 4个 ,编码区 1个 ,3′非编码区 7个。其中一个SNP能引起IL 1R1跨膜区氨基酸的替代 ,生物信息学分析显示它能够引起跨膜区结构的改变。结论IL 1R1基因变异可能对IL 1R1的功能产生影响。     

Deletion of the C-terminal end of aspartylgiucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement

Aspartyiglucosaminuria (AGU) is an inborn error of glycoproteincatabolism and represents the only known human deficiency ofan amidase, aspartyiglucosaminidase (AGA, EC 3.5.1.26 [EC] ). We reporthere a detailed characterization of a unique 2 kb deletion ofthe AGA gene in a North American AGU patient. To facilitatethe characterization of the deletion, genomic lamda clones spanningthe 3' flanking region of human AGA were isolated and sequenced.The breakpoint of the deletion was determined from the patient'sDNA by sequencing the genomic region containing the novel junction.The rearrangement involved a nonhomologous recombination withonly 2 bp of homology at the deletion breakpoint. The deletion's5' breakpoint was located in the last intron of AGA, thus abolishingthe normal C-terminal exon. This is in contrast to our previousfindings indicating that the deletion in the AGA gene wouldcontain only the complete 3' untranslated region and leave thecoding region intact (1). The unique feature of this deletionis a triplication of 19 thymidine nucleotides of an invertedAlu repeat, which is located at the deletion 3' breakpoint.The analysis of the patient's AGA cONA revealed an open readingframe containing a novel C-terminal exon, coding for a 64 aminoacid sequence, which has no homology to the normal exon 9 ofAGA. This new exon has a functional splice acceptor site atits 5' end, a stop codon, and a polyadenylation signal at the3' end. Expression of the mutant AGA cDNA in COS cells showedthat mutant mRNA is synthesized in equal amounts compared withnormal. However, the mutant polypeptide precursor is not processedinto the mature subunits of AGA, and is totally degraded within24 h of synthesis.     

Cloning of a Salmo salar interleukin-1 receptor-like cDNA

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily, defined by a cytosolic Toll/IL-1R (TIR) signalling domain, participates in host responses to injury and infection. We describe in this study the cloning of a cDNA encoding a Salmo salar interleukin-1 receptor-like protein (SalIL-1RLP). SalIL-1RLP comprises a potential signal peptide, three extracellular immunoglobulin domains, a short transmembrane region and an intracellular region that contains the TIR domain. The predicted amino acid sequence of SalIL-1RLP displays 43-44% similarities and 31% identities to chicken and human IL-1RI sequences. Within the intracellular region, SalIL-1RLP displays highest similarity (59%) and identity (46%) to the chicken IL-1RI sequence. Two different 5' distal UTRs were identified among six salmon IL-1RLP clones. The six clones, however, displayed identical 5' proximal UTRs, coding regions and 3' UTRs. SalIL-1RLP expression is induced in liver, head kidney, spleen and gills upon injection of salmon with bacterial lipopolysaccharide. Sequence comparisons, protein domain structures, expression patterns and phylogenetic analyses indicate that SalIL-1RLP is most closely related to type I interleukin-1 receptors and interleukin-1 receptor related proteins.