The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23)

Recently, we proved the existence of the second calmodulin family protein in Tetrahymena (Tetrahymena calcium-binding protein of 25 kDa, TCBP-25) by analyzing its cDNA (Takemasa, T., Ohnishi, K., Kobayashi, T., Takagi, T., Konishi, K., and Watanabe, Y. (1989) J. Biol. Chem. 264, 19293-19301). During the amino acid sequence determination of TCBP-25, we became aware of the fact that another polypeptide carrying calcium-binding domains of EF-hand type existed in addition to Tetrahymena calmodulin and TCBP-25. This third calmodulin family protein from Tetrahymena was confirmed by isolating its cDNA clones. One of the cloned cDNAs contains 763 nucleotides and encodes a protein that is composed of 207 amino acid residues and has a molecular mass of 23,413 daltons. This predicted protein possesses four EF-hand type calcium-binding domains, so we have designated it as Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). TCBP-23 is similar (35% homology) but clearly different from TCBP-25. The TCBP-23 gene is actively transcribed in vivo as a 0.84-kilobase RNA. Thus, it follows that Tetrahymena cells have three different calmodulin family proteins: calmodulin, TCBP-25 and TCBP-23. These proteins are expected to provide important clues for solving the mechanisms of calcium-dependent phenomena, such as ciliary reversal.     

Amino acid sequence of a calcium-binding protein (TCBP-10) from Tetrahymena

The amino acid sequence of a calcium-binding protein obtained from the cilium and cell body of Tetrahymena, designated as TCBP-10 (Tetrahymena calcium-binding protein; molecular mass = 10 kDa [Ohnishi, K. and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985] was determined. It is composed of 102 amino acid residues. The exact molecular mass is calculated to be 11563 Da. From the amino acid sequence analysis, it has two EF-hand-type calcium-binding sites.     

Cloning and sequencing of the gene for a 120-kDa immunodominant protein of Ehrlichia chaffeensis

Ehrlichia chaffeensis is the tick-borne, obligately intracellular bacterium that causes human monocytic ehrlichiosis. A 120-kDa protein is one of the immunodominant proteins of E. chaffeensis that stimulates production of specific antibodies in infected humans. A genomic library of E. chaffeensis was constructed in a λZAP II phage vector, and a clone expressing the 120-kDa protein of E. chaffeensis was identified using canine anti-E. chaffeensis serum. DNA sequence analysis of the cloned 120-kDa protein gene of E. chaffeensis identified a 1884-bp open reading frame with an ehrlichial promoter. Five identical 240-bp tandem repeat units were identified in the 120-kDa protein gene of E. chaffeensis, comprising 60% of the entire gene. Aside from the first repeat unit, all the other repeat units are identical. In the first repeat unit there are four nucleotides that are different from the other repeats. Hydropathy analysis of the deduced amino-acid sequence demonstrated that the repeat domain contains highly hydrophilic segments. The 120-kDa protein should be evaluated for a role in stimulating protective immunity.     

Tetrahymena actin. Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.     

Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.

p24 is a thyroid protein (Mr 24,000) identified by two-dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up-regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target-site for phosphorylation by cyclic AMP-dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through 'EF hand' domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+-phosphatidylinositol cascades.     

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum

Abstract Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-β-d-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69 716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKTIO) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.     

幽门螺杆菌外膜蛋白25基因的克隆及序列分析

目的 克隆幽门螺杆菌（Helicobacter pylori,Hp）外膜蛋白25（OMP25）基因,并对其进行序列分析。方法 利用PCR技术扩增OMP25基因,并将其定向插入pET-22b（＋）载体中,以DNA自动序列分析仪进行核苷酸分析。结果 DNA序列分析表明,所克隆的OMP25基因序列与GeneBank公布的一致。结论 该研究获得了序列正确的幽门螺杆菌OMP25基因,为其重组表达及其棚关研究奠定了良好基础。     

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.     

Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M_r of the mature polypeptide was 15674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.     

Cloning and sequencing of biofilm-associated protein (bapA) gene and its occurrence in different serotypes of Salmonella

Aims: Salmonella spp. has the capability to form biofilm on various surfaces. Biofilm‐associated protein (bapA), a large surface protein has been shown to play a leading role in the development of biofilm in Salmonella. Objective of this study was to investigate the presence of bapA gene in different serotypes of Salmonella spp. and to characterize DNA fragment encoding bapA protein of Salmonella Enteritidis. Methods and Results: Sixty‐seven Salmonella strains belonging to 34 serovars isolated from diverse sources in India were screened for the presence of bapA gene employing a primer designed for the purpose. All the strains yielded a positive amplification indicating that the bapA gene is well conserved in Salmonella spp. The amplified gene fragment of bapA was cloned in Escherichia coli (DH5 α) cells by using pGEM‐T easy cloning vector. On partial sequence analysis, the product exhibited 667 base pairs, corresponding to 218 amino acids. Conclusions BapA gene was found to be highly conserved in Salmonella. Partial sequence analysis of this gene from a strain of Salm. Enteritidis revealed close association with serotypes of poultry origin and also with some other animal/zoonotic serotypes. Significance and Impact of the Study: BapA gene can be targeted for the genus‐specific detection of this organism from different sources. Antigenic index of bapA protein indicates its protective and diagnostic potentials.     

Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein.

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Structural analysis of a developmentally regulated 25-kDa protein gene of Sarcophaga peregrina

In the previous paper, we described the identification of two abundant mRNAs of Sarcophaga peregrina (flesh-fly) which are selectively expressed in the fat body of middle third instar larvae. One of these mRNAs was found to encode a protein with a molecular mass of about 25,000 (25-kDa protein) when translated in vitro (Tamura, H., et al. (1983) Dev. Biol. 99, 145-151). Present paper reports the nucleotide sequence of a 2.3 kb DNA containing the entire gene for the 25-kDa protein. This gene consisted of four exons and contained an open reading frame for 184 amino acids. A CAT box and a TATA box were found in the 5'-flanking sequence. A poly A addition signal of AATAAA was assigned to the non-coding region in the fourth exon. A sequence having 75% homology with SV40 enhancer core sequence was identified in the non-coding region of the first exon.