人溶菌酶基因的合成和克隆

用固相亚磷酰胺法合成了人溶菌酶的全基因,全长为409bp,它包括了编码人溶菌酶的结梅基因,起始密码于ATG,终止密码子TAA、TGA,以及两端的BamHI和SphI的识别顺序。整个基因分成24个寡聚核苷酸片段进行合成,每个片段长度分别为26至38个核苷酸．然后用两种方法酶促连接成完整的人溶菌酶基因。基因克隆到M13载体上。用点杂交和限制酶酶切分析确定阳性克隆株。用双脱氧链终止法进行序列分析,证实所合成的人溶菌酶基因序列与设计的完全一致。     

Euglena gracilis chloroplast transfer RNA transcription units. II. Nucleotide sequence analysis of a tRNAVal-tRNAAsn-tRNAArg-tRNALeu gene cluster

The tRNA-coding locus of the 8.2-kilobase pair (kbp) Eco RI fragments Eco G of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA was chosen for detailed analysis. Two recombinant plasmids, pPG14, containing Eco G and the vector pMB9, and pEZC23, containing the chloroplast DNA fragment HindIII B cloned in pBR322 were employed for the study. The tRNA locus was mapped to an 0.8-kbp region of Eco G also present in HindIII B. The DNA sequence of a 1.6 kbp from HindIII B, containing the entire tRNA gene locus was determined. Four tRNA genes were identified from the DNA sequence. The gene organization is tRNAVal-16 bp spacer-tRNAAsn-3 bp spacer-tRNAArg-45 bp spacer-tRNALeu. The tRNALeu gene is of the opposite polarity as the other three genes. This is the first evidence of such a tRNA cluster for a chloroplast genome. Also evident from the DNA sequence, 132 bp from the 5'-end of the tRNALeu gene, is a putative gene or pseudogene for a chloroplast protein.     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

Isolation of Pollutant (Pine Needle Ash)-Responding Genes from Tissues of the Seaweed Ulva Pertusa

Genetic responses of the seaweed Ulva pertusa to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues treated in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responding to the stress were selectively isolated from agarose gel and sequenced with a DNA auto sequencer. According to sequence analysis, an ash-inducible gene (342 bp) and an ash-suppressed gene (1690 bp) were identified as hypothetical proteins.     

Molecular characterization of lysozyme type II gene in rainbow trout (Oncorhynchus mykiss): evidence of gene duplication

Rainbow trout (Oncorhynchus mykiss) have two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme type II appears to be a candidate gene for enhancing disease resistance in fish as well as livestock species. Using polymerase chain reaction the lysozyme type II gene was amplified from genomic DNA isolated from rainbow trout. Two amplified fragments of 2041 and 2589 bp were observed. Sequencing revealed both amplicons were lysozyme genes having nearly identical nucleotide sequences, except the longer fragment has 548 base pairs inserted in intron 2 at nucleotide position 513 and a few point mutations within intron 2. Both versions of trout lysozyme type II gene were comprised of four exons and three introns. We also demonstrated that trout lysozyme is most likely encoded by these two different genes.     

Hierarchical strategy for protein folding and design: synthesis and expression of T4 lysozyme gene and two putative folding mutants

A T4 lysozyme-coding DNA sequence of 495 bp was chemically synthesized and cloned by ligation of 26 deoxyribooligonucleotide fragments in two steps with a linearized plasmid followed by transformation. On selection by colony hybridization and DNA sequence analysis, clone pTLY.10 was identified to contain a complete T4 lysozyme synthetic DNA. On expression under lac-promoter, unfused T4 lysozyme was obtained in approximately 4-6% yield. The design and synthesis of two putative folding mutants, flexible (Gly-Gly-Gly) and rigid (Asn-Asp-Gly) at position 73-74-75, were based on hierarchical principles. Both mutants lost enzymatic activity of the wildtype. These results are readily understandable if the hierarchical organization of the structure is taken into account. A possible explanation is that the catalytic sites are blocked in both mutants.     

The nucleotide sequence of repetitive monkey DNA found in defective simian virus 40.

DNA fragments containing monkey DNA sequences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was determined using both RNA and DNA sequencing methods. The nucleotide sequence of this homogeneous DNA segment does not contain discernible multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration frequency of the sequence in the monkey genome is >10⁶. Digestion of total monkey DNA (from uninfected cells) with endonuclease R Hind III produces relatively large amounts of discrete DNA fragments that contain extensive regions homologous to the fragment isolated from the defective SV40 DNA.A second fragment, also containing monkey sequences, was isolated from the same defective substituted SV40 genome. The nucleotide sequence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined.The sequences in both fragments are also present in other, independently derived, defective substituted SV40 genomes.     

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts

Abstract Aspergillus oryzae , which is widely used for Japanese traditional fermentation, produced at least two lipolytic enzymes (L1 and L2). Southern hybridization analysis of restriction enzyme-digested genomic DNA fragments of Aspergillus oryzae with 23-mer oligonucleotides synthesized according to the amino acid sequence of the enzyme L1 as probes suggested that there is single copy of the L1 gene in the genome. DNA fragments containing the L1 gene were cloned in Escherichia coli . Nucleotide sequencing of the DNA fragments revealed an open reading frame consisting of 213 amino acid residues. It had three putative introns whose sizes were 52 bp, 48 bp and 53 bp, respectively. Putative CAAT and TATA boxes were found at positions −147 and −100 from A (+1) of the translational initiation codon, and a polyadenylation site at 158 bp downstream of the stop codon. The deduced amino acid sequence of the L1 gene was highly similar to those of cutinases from phytopathogenic fungi. Thus, it is interesting to note that the non-phytopathogenic fungus, A. oryzae , produces cutinase, which seems to play an important role in flavor formation.     

人恶性疟杂合多肽抗原基因化学合成及克隆

本文报道用固相亚磷酰胺法合成人恶性疟杂合多肽抗原基因。基因全长为216bp,分为10个寡聚核苷酸片段分别合成,然后经T4 DNA连接酶按设计顺序连接成完整的杂合抗原基因,重组到噬茵体M13 mp 18 RF DNA内,转染大肠杆菌JM109。用分子杂交和酶切分析筛选出重组克隆体。经序列分析,证明所合成的人恶性疟杂合多肽抗原基因与所设计完全一致。     

Multiplex polymerase chain reaction method discriminating <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Shigella</Emphasis> sp.

To distinguish between Escherichia coli and other bacteria that have similar biochemical characteristics, 3 polymerase chain reaction techniques were combined. The primer sets cydA-F2-A2 and cydA-R2-A2 were designed to amplify 605 base pairs of nucleotide sequence specific for the cydA gene of Escherichia coli; primer sets lacZ-F-A and lacZ-R-A to amplify 1,023 bp of nucleotide sequence specific for the lacZ gene of Escherichia coli; and primers lacA-F2-A2 and lacA-R2-A2 to amplify 325 bp of nucleotide sequence specific for the lacA gene of Escherichia coli. As a result, 3 nucleotide fragments were generated when 3 samples DNA from Escherichia coli were used as template. On the other hand, 1,023- and 605-bp products were obtained when DNA of Shigella sonnei was used, and a 605-bp product was obtained when DNA of Shigella flexneri was used. The specificity of the technique was confirmed by comparing it with the conventional culture test; the consistency rate of both tests was 0.749. These results suggest that the technique described in the present study will be useful for distinguishing Escherichia coli from Shigella species with accuracy and specificity.     

Cloning and nucleotide sequencing of the tyrosine phenol lyase gene fromEscherichia intermedia

Summary The tyrosine phenol lyase (TPL) gene was cloned from the genomic DNA of aEscherichia intermedia strain and the nucleotide sequence of the TPL structural gene was determined. The 1801 bpHincll-Nrul DNA fragment containing the TPL gene had an open reading frame of 1368 bp and the deduced amino acid sequence was 456 residues long with a molecular weight of 51,441 daltons.     

The 2005 version of the chloroplast DNA sequence from tobacco (Nicotiana tabacum)

The complete nucleotide sequence of tobacco chloroplast DNA was first determined in 1986, and then its updated gene map was reported in 1998. During the course of sequencing the chloroplast DNA ofNicotiana sylvestris, the female progenitor of tobacco, we found some sequence errors and amended the 1998 version. The tobacco chloroplast DNA comprises 155,943 bp, 4 bp longer than the 1998 version.     

Germ line-restricted,highly repeated DNA sequences and their chromosomal localization in a Japanese hagfish (Eptatretus okinoseanus)

The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.by W. Hennig     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Summary A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.     

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.     

Isolation of Plant Gene Space-Related Sequence Elements by High C+G Patch (HCGP) Filtration: Model Study on Rice

For the isolation of gene space representative sequence elements, a new methodology—high C+G patch (HCGP) filtration—has been developed using rice as a model. The method is based on the fragmentation of the genomic DNA by methylation-sensitive HpaII and MspI restriction endonucleases having exclusively G/C base pair-containing recognition sites. These enzymes fragment the genome at high C+G content and hypomethylated regions. Cloning fragments spanning such regions in close vicinity (200–2,000 bp) revealed that about 60% of the clones represented gene space sequences resulting in twofold enrichment of these sequences, which is close to the theoretical maximum in rice. The sequence information of clones used in the present study was deposited in the NCBI database under the accession numbers EI 365676–EI 366364.