Cloning and Nucleotide Sequencing of the Antitumor Antibiotic C-1027 Apoprotein Gene

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met¹—Ala³³ leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, ¹⁾ (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mernA) while the flanking regions had little homology with the corresponding ones of mernA, except some homology at the – 10th and – 35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.     

Molecular cloning and sequence determination of the tuf gene coding for the elongation factor Tu of Thermus thermophilus HB8

The tuf gene, which encodes the elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, and its flanking regions were cloned and sequenced. The gene encoding EF-G was found upstream of the 5' end of the tuf gene. The tuf gene of T. thermophilus HB8 had a very high G + C content and 84.5% of the third base in codon usage was either G or C. The deduced primary structure of the EF-Tu was composed of 405 amino acid residues with a Mr = 44658. A comparison of the amino acid sequence of EF-Tu from T. thermophilus HB8 with those of Escherichia coli and Saccharomyces cerevisiae mitochondria showed a very high sequence homology (65-70%). Two Cys residues out of the three found in E. coli EF-Tu had been replaced with Val in T. thermophilus HB8 EF-Tu. An extra amino acid sequence of ten residues, consisting predominantly of basic amino acids (Met-182-Gly-191), which does not occur in EF-Tu of E. coli, was found in T. thermophilus HB8.     

Bacillus megaterium spore protein C-3: nucleotide sequence of its gene and the amino acid sequence at its spore protease cleavage site

The nucleotide sequence of the Bacillus megaterium gene coding for spore-specific protein C-3 has been determined. The gene codes for 65 amino acids and the coding sequence is preceded by an efficient ribosome-binding site. The predicted protein C-3 sequence agrees with both the amino acid composition and the amino terminal sequence of protein C-3, and shows homology (approx. 65 % of all residues are identical) with the sequences of the analogous proteins A and C of B. megaterium. Protein C-3 is cleaved by the sequence-specific B. megaterium spore protease, and the amino acid sequence at the new amino-terminus generated is identical to that predicted from the gene sequence, and homologous to the spore protease cleavage sites in the A and C proteins. The protein C-3 gene also shares a number of features with the previously sequenced protein C gene in both upstream and downstream flanking sequence.     

转座子引起的猪ktn1基因结构变异及其与生产性能的关联分析

为探明转座子对猪的ktn1基因及其侧翼区结构变异的贡献,从全基因组测序(WGS)数据库中获取14个猪基因组中的ktn1基因序列和侧翼序列,通过ClustalX多序列比对和RepeatMasker转座子注释,全面解析转座子对ktn1的影响。通过PCR检测到一个SINEA1转座子插入多态,在苏姜猪群体中与相关性状进行关联分析。结果显示,ktn1基因及其侧翼区中含有至少77个转座子片段,其中绝大部分(98.70%)为SINE类转座子,并鉴定到9个小结构变异和4个由转座子引起的大结构变异,表明转座子是基因变异的重要来源。其中一个SINEA1插入多态引起的结构变异,在不同品种中呈现丰富的多态性,且无插入个体(SINE-/-)苏姜猪的断奶窝重((64.20±10.6) kg)比纯合有插入个体(SINE+/+)((74.14±9.0) kg)和杂合有插入个体(SINE+/-)((69.71±7.7) kg)轻(P<0.05),表明基于转座子插入多态研发分子标记具有可行性,提示转座子插入多态分子标记在分子辅助育种中具有较强的应用潜力。     

Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore

C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The three-dimensional solution structures of the 110 residue (10.5 kDa) C-1027 apoprotein and its complex with the aromatized chromophore have been determined separately by homonuclear two-dimensional nuclear magnetic resonance methods. The apoprotein is mainly composed of three antiparallel beta-sheets: four-stranded beta-sheet (43-45, 52-54; 30-38; 92-94; 104-106), three-stranded beta-sheet (4-6; 17-22; 61-66), and two-stranded beta-sheet (70-72; 83-85). The overall structure of the apoprotein is very similar to those of other chromoprotein apoproteins, such as neocarzinostatin and kedarcidin. A hydrophobic pocket with approximate dimensions of 14 A x 12 A x 8 A is formed by the four-stranded beta-sheet and the three loops (39-42; 75-79; 97-100). The holoprotein (complex form with the aromatized chromophore) structure reveals that the aromatized chromophore is bound to the hydrophobic pocket found in the apoprotein. The benzodihydropentalene core of the chromophore is located in the center of the pocket and other substituents (beta-tyrosine, benzoxazine, and aminosugar moieties) are arranged around the core. Major binding interactions between the apoprotein and the chromophore are likely the hydrophobic contacts between the core of the chromophore and the hydrophobic side-chains of the pocket-forming residues, which is supplemented by salt bridges and/or hydrogen bonds. Based on the holoprotein structure, we propose possible mechanisms for the stabilization and the release of chromophore by the apoprotein.     

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.     

Nucleotide sequence of a secondary attachment site for bacteriophage lambda on the Escherichia coli chromosome.

The nucleotide sequence of a secondary attachment site for bacteriophage lambda was determined in a region near the rrnB gene at 88 min on the E. coli chromosome. The sequence has a 8 base pair interrupted homology GCT TTTTA to the common core of the primary attachment site (attB) and the corresponding phage sequence (attP). The site of crossover during integration lies probably between nucleotides -3 and +1. The flanking regions have no obvious homology to the arms of either attP or attB.     

Evidence for the classification of a crayfish pathogen as a member of the genus Coxiella

AIMS: The study aimed to provide characterization of a potential new species of Coxiella, identified following a series of outbreaks of disease in Australian native freshwater crayfish. METHODS AND RESULTS: PCR primers designed for amplification of Coxiella burnetii genes including 16S rDNA, com1 and sodB were used to amplify homologues in the Coxiella-like crayfish pathogen. Products were then cloned and sequenced. The organism demonstrated a high degree of sequence homology in the highly conserved 16S rDNA (96%) and sodB (99%) genes, as well as the Coxiella sp. specific com1 (100%) gene. Regions flanking the sodB coding sequence demonstrated homology to C. burnetii antioxidant AhpC/Tsa family protein and dihydrodipicolinate reductase gene. CONCLUSIONS: The degree of homology between the genes selected and flanking regions suggested the two organisms were sufficiently closely related to belong to the same genus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided evidence for a potential new species in the currently monospecific genus Coxiella, with the only described member being C. burnetii, a category B biological warfare agent.     

Drosophila melanogaster U1 and U2 small nuclear RNA genes contain common flanking sequences

The flanking sequences of three U2 genes (or pseudogenes) and one U1 gene of Drosophila melanogaster have been determined. Comparison of the sequences reveals a remarkable homology between position ?30 and ?65 upstream from the structural genes, starting with a TATA box-like sequence. The 3′ flanking regions are also conserved in all genes and contain a canonical A-A-T-A-A-A polyadenylation signal.     

ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.