Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.     

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.     

Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium.

Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAP1; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated map1, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium.     

Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the matrix protein

The sequence of the matrix (M) protein gene and contiguous intergenic regions of the human parainfluenza 3 virus (PF3) was determined by molecular cloning. The encoded M protein contains 354 amino acids and has a predicted mol wt of 39,506. The M protein amino acid sequence was compared to the homologous proteins from other members of the Paramyxoviridae family. The PF3 protein shared 61 % homology with the Sendai virus protein and approximately 35% homology with measles and canine distemper virus proteins. Little homology was observed with respiratory syncytial virus. The M protein appears to be the most highly conserved among the Paramyxoviridae proteins.     

Cloning and nucleotide sequence analysis of aBrucella abortusgene encoding an 18 kDa immunoreactive protein

A DNA fragment encoding an 18 kDa protein fromBrucella abortusstrain 2308 was cloned and expressed inEscherichia coliThis recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an 87 kbpSau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an 30 kbpPstI DNA fragment DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17 529 Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) fromB abortusstrain 544 designated Omp19.     

Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein

We cloned the full-length cDNAs corresponding to the mRNA for the hemagglutinin (H) protein of rinderpest virus (RV) and determined the nucleotide sequence of RV-H. The gene of RV-H was composed of 1952 nucleotides and contained a single large open reading frame, which was capable of encoding a protein of 609 amino acids with a molecular weight of 68,330 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of the measles virus (MV)-H. The 5' end of the message (nucleotides 1 to 485) was largely conserved, with a homology of 75.1% of the nucleotides and 78.0% of the predicted amino acids. In the middle portion (nucleotides 486-1310), where the potential glycosylation sites exist, 56.6% of the nucleotides and 49.5% of the amino acids were identical. In the 3' end of the message (nucleotides 1311-1850), 63.3% of the nucleotides and 58.1% of the amino acids were identical. Four potential glycosylation sites were found in RV-H protein and three of them were the same as those of MV-H protein. The positions of 13 cysteine residues of RV-H were absolutely identical to those of MV-H. The hydropathy profile of RV-H protein resembled that of MV-H. One major hydrophobic region long enough to be an anchor in the membrane was located near the N-terminus.     

Molecular cloning,sequence analysis and expression of the gene encoding an antifungal-protein from Aspergillus giganteus

The gene encoding the precusor of a small secretory protein with antifungal activity was isolated from A. giganteus and characterized by restriction mapping, hybridization and nucleotide sequencing. The promoter contains a typical TATA-box at a distance of 135 bp upstream of the open reading frame. The open reading frame is interrupted by two small introns with conserved splice sites. The precursor of the antifungal protein (AFP) consists of 94 amino acids and appears to be processed to the mature AFP of 51 amino acids by a two-step process. Transfer of the gene into A. niger yielded only transformants with a very low expression level, probably because high-expression transformants were counterselected by the antifungal activity of the recombinant protein.     

Molecular cloning and nucleotide sequence of cDNA encoding a functional murine low-density-lipoprotein receptor

The low-density-lipoprotein (LDL) receptor is an important mediator of mammalian cholesterol metabolism; its congenital absence in humans is characterized by hypercholesterolemia, atherosclerosis, and coronary artery disease. We report here the identification and cloning of a cDNA encoding the murine LDL receptor. The cDNA insert is 4467 base pairs in length and the deduced amino acid sequence bears 78.2% homology with the reported human sequence. This murine cDNA was subcloned into a retroviral-based expression vector, LmLSN1, and transfected into 3T3 cells. The production of functional LDL receptor was confirmed by ligand binding of DiI-LDL cholesterol.Sequence data from this article has been deposited with the EMBL/GenBank Libraries under Accession No. X64414.     

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.     

Molecular cloning and sequence analysis of the scpZ gene encoding the serine carboxypeptidase of Absidia zychae

Carboxypeptidase Z is a serine carboxypeptidase secreted by Absidia zychae NRIC 1199. The cDNA and genomic DNA carrying the scpZ gene encoding carboxypeptidase Z were cloned and sequenced. The nucleotide sequences of the cDNA (1.4 kb) and the genomic DNA (3.3 kb) were analyzed and the intervening sequences were located by a comparison of the two. It was found that the scpZ gene was interrupted by 11 short introns, 50–75 nucleotides in length. Genomic Southern analysis showed that there was only one scpZ gene in the genome of A. zychae. The gene encoded a putative pre-proenzyme composed of 409 amino-acid residues of the mature carboxypeptidase Z (Mr 45 421) and an additional N-terminal sequence of 51 amino-acid residues. The amino-acid sequence around the active serine residue of carboxypeptidase Z (-G-E-S-Y-G-G-) differed from the consensus (-G-E-S-Y-A-G-) which is conserved in most of the serine carboxypeptidases so far analyzed.     

Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein

The nucleotide sequence of the gene encoding the major nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) has been determined. The N mRNA is 1196 nucleotides long with a single, large open reading frame. The derived polypeptide has 391 amino acids corresponding to a calculated molecular weight of 42,600 Da. This is in agreement with the molecular weight of 43,000 Da determined for the BRSV N protein by SDS-polyacrylamide gel electrophoresis (PAGE). Comparison of the nucleotide sequence of BRSV N gene with the sequence of the N gene of human respiratory syncytial virus (HRSV) revealed a homology of 80.7%. There is a 93.3% homology at the amino acid level between the N proteins of BRSV and HRSV. The 5'- and 3'-terminal untranslated sequences that are conserved among HRSV mRNAs were also identified in the N mRNA of BRSV. The results indicate that the N genes are highly conserved in the bovine and human strains of respiratory syncytial virus.     

Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus.

Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an apparent molecular weight of 40,000. The nucleotide sequence of the entire DNA insert was determined. Two open reading frames coding for polypeptides with calculated molecular weights of 21,893 and 30,721 were identified. The larger open reading frame encoded LppB, while the smaller reading frame encoded a nonseroreactive protein with a relative molecular mass of approximately 18 kDa. The 16 amino-terminal amino acids of the deduced LppB polypeptide showed strong sequence homology to the signal peptide of secreted bacterial proteins, and the sequence Leu-Ala-Ala-Cys at the putative cleavage site corresponds to the consensus cleavage sequence of bacterial lipoproteins. Synthesis of the mature LppB lipoprotein in H. somnus was inhibited by globomycin, a specific inhibitor of signal peptidase II. LppB was localized to the outer membrane of H. somnus.     

Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence

The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.     

Cloning, nucleotide sequence, and expression of the gene coding for a ribosome releasing factor-homologous protein of Brucella melitensis.

The gene coding for a Brucella melitensis cytosoluble protein (CP24) that is immunogenic in infected sheep and a major component of brucellin INRA was cloned and sequenced. As in Brucella cells, CP24 was located in the cytoplasm of recombinant Escherichia coli. The amino acid sequence predicted from the cloned gene revealed 48 and 46% identity with the ribosome releasing factor, a protein factor required for release of the 70S ribosome from the mRNA, of E. coli and Haemophilus influenzae Rd, respectively. Sera from naturally infected sheep and sheep experimentally infected with B. melitensis H38 showed antibody reactivity against recombinant CP24.