Identification and isolation of Cryptosporidium parvum genes encoding microtubule and microfilament proteins.

Microtubules and microfilaments are highly conserved cytoskeletal polymers hypothesized to play essential biomechanical roles in the unusual gliding motility of Apicomplexan zoites and in their invasion of, and development within, host epithelial cells. We have identified and isolated Cryptosporidium parvum genes encoding the microtubule proteins alpha- and beta-tubulin and the microfilament protein actin by screening a lambda gt11 C. parvum genomic DNA library with degenerate oligonucleotide and heterologous cDNA hybridization probes respectively. The alpha- and beta-tubulin genes have been partially sequenced and the deduced peptide sequences show greatest homology with the tubulins of the related parasites, T. gondii and P. falciparum. The complete nucleic acid sequence of the actin gene predicts a 376 amino acid, 42 kDa protein having 85% sequence identity with the P. falciparum actin I and the human gamma-actin proteins. Each of these cytoskeletal protein genes was demonstrated to be of cryptosporidial origin by Southern analyses of C. parvum chromosomes fractionated by pulsed field gel electrophoresis; the cloned alpha- and beta-tubulin genes hybridized with chromosomes of ca. 1,200 and 1,500 kb respectively and the cloned actin gene also hybridized with a 1,200 kb chromosome.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

Structure of the 3' portion of the bovine elastin gene

A bovine genomic library constructed by partial Sau3A digestion and contained in lambda Charon 30 was screened by in situ hybridization with a 1.3-kilobase (kb) sheep elastin cDNA clone [Yoon, K., May, M., Goldstein, N., Indik, Z., Oliver, L., Boyd, C., & Rosenbloom, J. (1984) Biochem. Biophys. Res. Commun. 118, 261-269]. Three clones encompassing 10 kb of the bovine elastin gene were identified and characterized by restriction mapping and DNA sequencing of the 6.2 kb of the most 3' region of the gene. These analyses have permitted localization of eight exons in the 6.2 kb in which the translated exons vary in size from 27 to 69 base pairs, and there is an approximately 1-kb untranslated region at the 3' end. In addition to identification of sequences homologous to those found in porcine tropoelastin, the analyses defined a 58 amino acid sequence that forms the carboxy-terminal region of tropoelastin, and this sequence, which contains two cysteine residues, was previously not observed in the protein sequence data. The analyses also suggest that functionally distinct cross-link and hydrophobic domains of the protein are encoded in separate exons.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

柔嫩艾美耳球虫孢子化卵囊cDNA文库的构建

用建立表达性文库的方法 ,构建了Eimeriatenella孢子化卵囊噬菌体ZAP表达性cDNA文库。首先用TRIzol试剂盒从E .tenella孢子化卵囊中提取总RNA ,再用Oligo(dT)₁₂纤维素柱从总RNA中分离mRNA ,以mRNA为模板 ,Oligo(dT)₁₈Linker Primer为引物 ,反转录合成cDNA第一链 ,再在DNA聚合酶Ⅰ作用下置换合成第二链cDNA。cDNA第二链合成后用PfuDNA聚合酶补平XhoⅠ位点 ,再与EcoRⅠAdapters连接 ,经XhoⅠ酶切后 ,凝胶电泳回收 500bp～4.0kp之间的cDNA片段 ,纯化后的双链cDNA与载体ZAPExpressvector连接。体外包装后得到E .tenella孢子化卵囊的cDNA表达性文库 ,经测定该文库的容量为 6×10⁶ ,扩增后文库的滴度为 1×10¹¹ Pfu mL ,经PCR测定 ,该文库的重组率为 96%。     

The cDNA of the two isoforms of bovine cGMP-dependent protein kinase

cDNAs encoding the isoform I alpha of the cGMP-dependent protein kinase were isolated from a bovine trachea smooth muscle cDNA library constructed in lambda gt10. The deduced protein sequence is identical with the protein sequence obtained by Edman degradation of the bovine lung enzyme [(1984) Biochemistry 23, 4207-4218]. Alternate cDNA clones were isolated which code for a protein slightly different within the aminoterminal part from the known amino acid sequence. These alternate cDNAs contain the sequence of a peptide identified in the isoform I beta of cGMP-dependent protein kinase. Northern blot analysis of poly(A)+ RNA from bovine trachea smooth muscle indicated the presence of two different mRNA species of about 6.2 kb.     

Sequence repetition and genomic distribution of small polydisperse circular DNA purified from HeLa cells

Covalently closed circular DNA molecules (cccDNA) from the human HeLa cell line were purified (96% pure by weight) by use of ATP-dependent deoxyribonuclease, and cloned into the HindIII site of phage lambda vector Charon 7. From the cccDNA library thus obtained, nine recombinants carrying mitochondrial DNA and 36 recombinants carrying small polydisperse circular (spc) DNA were picked at random for subsequent tests. The inserted fragments of spcDNA ranged in size from 0.6 to 7.6 kb with a mean length of 1.9 kb, a value which is the same as the average length of spcDNA. Analysis of the cloned spcDNA fragments revealed that (a) all the spcDNA clones investigated shared homologies with chromosomal DNA sequences, (b) all but one cloned DNA contained repetitive sequences, (c) the sequence organization could be roughly classified according to the reiteration frequency as greater than 10(5) (Alu family class), 10(4) to 10(5) (KpnI family class), 10(3) to 10(4) (mitochondrial DNA class) and less than 10(3) times per haploid genome, and (d) most of the repetitive sequences were dispersed in the genome, although some appeared clustered.     

Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1.5 kb labelled DNA probe containing the 3' region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8.5 kb Bg/II fragment containing the complete MOMP gene was cloned into lambda EMBL3. Two hybridizing EcoRI fragments were sub-cloned into the lambda ZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.     

Inhibition of topoisomerases from Pneumocystis carinii by aromatic dicationic molecules.

Pentamidine and related derivatives inhibit an ATP-dependent topoisomerase activity from Pneumocystis carinii extracts. Since it would be extremely difficult to purify ample quantities of the organisms to allow characterization of the enzyme and carry out drug binding experiments, we have begun the cloning of the topoisomerase genes with a goal towards expression of each gene in a heterologous system. Following construction of genomic libraries in the vectors lambda DASH and lambda ZAP, oligonucleotides corresponding to conserved regions of both topoisomerases I and II were used in the polymerase chain reaction (PCR) of P. carinii DNA to generate probes. Candidate clones for both genes have been identified. Partial DNA sequence of the topoisomerase II gene has been determined.     

Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome

A library of 900 recombinant phages has been constructed for the genome of Desulfovibrio vulgaris Hildenborough (1.7 x 10(6) bp) by cloning size-fractionated Sau3A fragments (15-20 kb) into the replacement vector lambda-2001. When a hydrogenase gene probe, a 4.7-kb SalI-EcoRI fragment of known nucleotide sequence, was used to screen the plaque lifted library, 23 positive clones were found, which together span 31 kb of D. vulgaris DNA. To facilitate the cloning of genes with oligodeoxynucleotides as probes, DNA was purified for all clones in the library and spotted on a 16 x 16-cm grid of nitrocellulose. This grid was incubated sequentially to identify lambda clones containing the gene for redox proteins of known amino acid sequence: cytochrome c3 (one 18-mer----four clones), flavodoxin (one 17-mer and one 26-mer----one clone) and rubredoxin (one 44-mer----21 clones). The four cyc-positive clones are also recognized by the rubredoxin oligodeoxynucleotide probe. Restriction mapping defines a 35-kb region of the D. vulgaris chromosome in which the rub and cyc loci are separated by 17.5 kb. The nucleotide sequence of the rubredoxin gene was determined and the deduced amino acid sequence found to agree with that determined in Bruschi [Biochim. Biophys. Acta 434 (1976) 4-17] with the exception of Thr-21 which is found to be encoded by GAC, an Asp codon. A plausible ribosome-binding site precedes the N-terminal initiator methionine residue. Rubredoxin does not have an N-terminal signal sequence which is in agreement with the cytoplasmic location of this redox protein.     

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.     

Osteonectin mRNA: distribution in normal and transformed cells.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.     

Isolation and fine structure organisation of an avian vitellogenin gene coding for the major estrogen-inducible mRNA

Two phage lambda recombinant DNA clones covering the entire sequence of an avian vitellogenin gene, plus flanking regions, have been isolated from an erythrocyte DNA gene library and characterized by R-loop and restriction mapping. The total length of this avian vitellogenin gene is 23 kb. The cloned sequences flanking the gene at the 5' and 3' end are 7 and 3 kb, respectively. The total length of exons in the two clones is 6.7 kb (vitellogenin mRNA is 6.6 kb). The gene is interrupted by at least 25 introns with a mean intron length of 940 bp. Some 6--10 additional very small introns may also be present but they were not observed reproducibly. The mean exon length is 250 bp. Restriction endonuclease digests of total liver genomic DNA and lambda recombinant DNA were also analyzed by electrophoresis. Southern blotting and hybridization with cloned vitellogenin cDNA. The results show an identity of organisation of this vitellogenin in the DNA from the two sources, thus ruling out a possible cloning artifact. In contrast to Xenopus vitellogenin we have found no evidence to suggest that avian vitellogenin is encoded by a small family of related genes.