Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Streptomyces albus G mutants defective in the SalGI restriction-modification system

Streptomyces albus G mutants (at least 12 of which were independent) defective in SalGI-mediated restriction (R-) were isolated after mutagenesis. Some of them lacked detectable SalGI activity in cell-free extracts. Some were also partially or completely defective in SalFI-associated modification (M-). Loss of restriction rendered S. albus G sensitive to many phages to which it was normally totally resistant. DNA from one such phage had many SalGI target sites (mean, one site per 1.35 kilobases). A mutant was isolated which was heat-sensitive for growth, apparently because it was restriction-proficient but temperature-sensitive for modification. At a rather high frequency, this mutant generated spontaneous heat-tolerant derivatives which were nearly all R-. Such R- mutants were always M- rather than being temperature-sensitive for modification. In a limited genetic analysis, the determinants of restriction and modification did not recombine with each other, and since there was no reassortment of these phenotypes among the parental output of crosses it appeared that the determinants were located close together on the chromosome.     

Characterization of a plasmid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.     

Organization of rRNA genes in Mycobacterium bovis BCG.

The number of rRNA genes in Mycobacterium bovis BCG was examined by Southern hybridization of end-labeled 5S, 16S, and 23S rRNAs with BamHI, PstI, and SalI digests of M. bovis BCG DNA. Each RNA probe gave only one radioactive band with three kinds of DNA digest. These results suggest that M. bovis BCG chromosomes may carry only a minimum set of rRNA genes. Hybridization of randomly labeled rRNAs with BamHI, PstI, SalI, BglII, and PvuII digests of DNA from the same organism supported these conclusions. The 6.4-kilobase-pair SalI fragment containing the entire structural genes for both 16S and 23S rRNAs was cloned into pBR322. The cloned fragment was characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis, and the R-loop technique. The results indicated that the fragments contained rRNA genes in the following order: 16S, 23S, and 5S rRNA genes. No tRNA gene was detected in the spacer region between the 16S and 23S rRNA genes, but one was found downstream of the 23S rRNA and 5S rRNA genes.     

The SalI (SalGI) restriction-modification system of Streptomyces albus G

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.     

Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant

The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E. coli PpcI genomic DNA. This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16. Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only. This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme. When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2. The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme. Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2.     

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle.

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.     

定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力

应用定向进化技术提高了嗜热拟青霉Paecilomyces thermophila J18耐热β-1,3-1,4-葡聚糖酶(PtLic16A)在酸性条件下的催化能力.结合易错PCR和DNA改组的方法,构建了β-葡聚糖酶的突变体文库;利用刚果红染色法建立了阳性克隆的高通量筛选体系.筛选得到的突变酶PtLic 16AM1的反应最适pH由7.0变化至5.5,且保持了原有的耐热性和比酶活.突变酶的DNA序列中有4个点位发生突变,引发了4处氨基酸替换,分别是T58S、Y110N、G195E和D221G.结构模拟结果显示,发生突变的4个氨基酸位点中,Y110N位置靠近酶活性中心,而T58S、G195E和D221G则离酶活性中心较远,其中T58S、G195E可能对酶最适pH的变化起到了关键作用.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Restriction of bacteriophage plaque formation in Streptomyces spp.

Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.     

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.     

Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli

Lederberg, Seymour (Brown University, Providence, R.I.). Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. J. Bacteriol. 91:1029-1036. 1966.-The locus for the host specific restriction and modification of deoxyribonucleic acid in Escherichia coli has been mapped by matings between mutants for these characters in strains K-12, C600, and B. Linkage analysis and kinetics of marker transfer indicate that a single or closely linked multiple chromosomal site located about 4 min counterclockwise to leucine is responsible for these activities. Secondary factors which affect the quantitative level of restriction also were detected. Wild-type recombinants were isolated in crosses between rm(-) (restriction or modification, or both) mutants. The expression in zygotes of the restrictionless character of a rm(-) donor is masked by a separate, physiological impairment of restriction, which results from mating and is independent of the modification state of the donor. The relevance of the restriction character to mating incompatibilities in these and other bacterial strains is considered.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization.

With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels. A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection. The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.     

Physical map of Campylobacter jejuni TGH9011 and localization of 10 genetic markers by use of pulsed-field gel electrophoresis.

The physical map of Campylobacter jejuni TGH9011 (ATCC 43430) was constructed by mapping the three restriction enzyme sites SacII (CCGCGG), SalI (GTCGAC), and SmaI (CCCGGG) on the genome of C. jejuni by using pulsed-field gel electrophoresis and Southern hybridization. A total of 25 restriction enzyme sites were mapped onto the C. jejuni chromosome. The size of the genome was reevaluated and was shown to be 1,812.5 kb. Ten C. jejuni genetic markers that have been isolated in our laboratory were mapped to specific restriction enzyme fragments. Furthermore, we have accurately mapped one of the three rRNA operons (rrnA) and have demonstrated a separation of the 16S and 23S rRNA-encoding sequences in one of the rRNA operons.     

Cloning of the ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) structural genes from Salmonella typhimurium LT2.

The structural genes of ADPglucose pyrophosphorylase (glgC) and glycogen synthase (glgA) from Salmonella typhimurium LT2 were cloned on a 5.8-kilobase-pair insert in the SalI site of pBR322. A single strand specific radioactive probe containing the N terminus of the Escherichia coli K-12 glgC gene in M13mp8 was used to hybridize against a S. typhimurium genomic library in lambda 1059. DNA from a plaque showing a positive hybridization signal was isolated, subcloned into pBR322, and transformed into E. coli K-12 RR1 and E. coli G6MD3 (a mutant with a deletion of the glg genes). Transformants were stained with iodine for the presence of glycogen. E. coli K-12 RR1 transformants stained dark brown, whereas G6MD3 transformants stained greenish yellow, and they both were shown to contain a 5.8-kilobase-pair insert in the SalI site of pBR322, designated pPL301. Enzyme assays of E. coli K-12 G6MD3 harboring pPL301 restored ADPglucose pyrophosphorylase and glycogen synthase activities. The specific activities of ADPglucose pyrophosphorylase and glycogen synthase in E. coli K-12 RR1(pPL301) were increased 6- to 7-fold and 13- to 15-fold, respectively. Immunological and kinetic studies showed that the expressed ADPglucose pyrophosphorylase activity in transformed E. coli K-12 G6MD3 cells was very similar to that of the wild-type enzyme.