Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Expression of human angiotensinogen cDNA in Escherichia coli

Human angiotensinogen cDNA clones were isolated from a human liver library. Nucleotide sequence analysis of these cDNA clones revealed that position 1075 in the messenger RNA, which is part of a PstI recognition sequence, is different from the published sequence (Kageyama, R., Ohkubo, H., and Nakanishi, S. (1984) Biochemistry 23, 3603-3609). This change results in an altered amino acid at this position in the corresponding protein sequence and suggests possible restriction fragment length polymorphism. The full length human angiotensinogen cDNA was constructed from partial cDNA clones and ligated into an isopropyl-1-thio-beta-D-galactopyranoside inducible bacterial expression vector pUC9 to develop expression plasmid pUCHAG27. This plasmid permitted the synthesis of human angiotensinogen in Escherichia coli. The recombinant bacteria overproduced a 53-kDa protein which was recognized by anti-human angiotensinogen antibodies. The synthesis of this protein was greatly increased upon induction with isopropyl-1-thio-beta-D-galactopyranoside. The chimeric protein, almost identical to human angiotensinogen, was partially purified by ammonium sulfate fractionation and gel filtration on Sephadex G-100. Human kidney renin was shown to enzymatically cleave this recombinant protein to produce des-(angiotensin I)-angiotensinogen and a small polypeptide. Thus, we provide evidence that recombinant human angiotensinogen synthesized through E. coli is biologically active and serves as a substrate for human renin.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene.

An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.     

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein

Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.     

Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin

An expression plasmid containing a hybrid gene encoding a protein having the primary amino acid sequence of the adenylate cyclase from Escherichia coli was constructed. When the gene was induced, the adenylate cyclase could be expressed at high levels in a cya- strain of E. coli. The majority of the enzymatic activity and protein (having a molecular weight of 95,000) induced was insoluble. However, treatment of the insoluble fraction of cell lysates with trypsin resulted in both an increase in and solubilization of the total amount of adenylate cyclase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the soluble protein produced by treatment with trypsin revealed a polypeptide having a molecular weight of 30,000. This soluble, catalytically active fragment of adenylate cyclase was purified and subjected to amino-terminal sequence analyses; two amino-terminal sequences were identified beginning at residue 82 and at residue 342 of the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified fragment followed by either silver or Coomassie Blue staining revealed the presence of only a single polypeptide having a molecular weight of 30,000; a short oligopeptide associated with the amino terminus at residue 342 could not be detected. Site-directed mutagenesis was used to place a stop codon at residue 341; the truncated enzyme was catalytically active, so the short oligopeptide is not necessary for catalysis. The Km for ATP, the Ka for Mg2+, and the Vmax determined for the product containing the 30,000-dalton fragment were similar to the values reported for the intact enzyme from E. coli.     

家蚕天蚕素cDNA原核表达及抗菌活性检测

采用RT-PCR方法从家蚕Bombyx mori新疆品种新蚕三号组织中扩增天蚕素cDNA片段,回收并克隆至Pmd18-T载体,进行序列分析。基因序列分析结果与已发表的天蚕素B的序列同源性为98%,表明所克隆的新疆家蚕天蚕素cDNA为独特的cDNA片段。将天蚕素基因与Pgex-4T-1融合表达载体中的谷胱甘肽转移酶基因融合,在大肠杆菌中表达, 结果表明经IPTG 诱导30 min后,pGEX-4T-1/天蚕素转化后的大肠杆菌生长明显受到抑制;当诱导210 min 后,大肠杆菌数量又开始增加,逐渐恢复至正常水平。说明天蚕素与谷胱甘肽转移酶基因融合表达后,在IPTG存在的短时间内仍然对原核细胞有较强的抗菌抑杀作用。     

Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Increased expression of a cloned gene by local mutagenesis of its promoter and ribosome binding site.

A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E. coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased beta-galactosidase synthesis, were selected for DNA sequencing. One clone has a G leads to A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C leads to T and T leads to C) between the Shine-Dalgarno sequence and initiation codon which raise expression of beta-galactosidase two-fold. A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present. These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.     

Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme

The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli. Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr. Although the C. pasteurianum and the E. coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes. Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E. coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences. A second clone containing this gene on a large restriction fragment was isolated by hybridization. This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase. Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene. Appropriate operon fusions with a promoter-less E. coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E. coli. The DNA sequence of this region which lies upstream of the C. pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase. The reasons for the high and low level expression and for the instability of the C. pasteurianum galactokinase in E. coli are discussed. The presence of the galactokinase suggests that galactose is used in C. pasteurianum through the Leloir pathway via galactose 1-phosphate.     

Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.