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K K Mark

Publications and source records attributed to K K Mark.

17 recordsLinked to original sources
PubMed1987

Occurrence of multiple antibiotic resistance and R-plasmids in gram-negative bacteria isolated from faecally contaminated fresh-water streams in Hong Kong.

The bacterial populations of six freshwater streams in populated areas of the Hong Kong New Territories were studied. There is considerable faecal contamination of these streams, with coliform counts as high as 10(5) c.f.u./ml and the contaminating organisms show a high prevalence of antibiotic resistance and multiple resistance. With direct plating of water samples onto antibiotic-containing media, an average of 49% of the gram-negative bacteria were ampicillin-resistant, 3% chloramphenicol-resistant and 1% gentamicin-resistant. At individual sites resistance to these drugs was as high as 98%, 8% and 3% respectively. More than 70% of strains were resistant to two or more antibiotics, 29% to five or more and 2% to eight or more. A total of 98 patterns of antibiotic resistance were detected with no one pattern predominating. Twenty-eight gram-negative bacterial species were identified as stream contaminants. Escherichia coli was the commonest bacterial species isolated and other frequent isolates were Enterobacter sp., Klebsiella sp. and Citrobacter sp., but no enteric pathogens were detected. The greatest prevalence of resistance and multiple resistance was associated with the heaviest contamination by E. coli. Analysis of selected stream isolates revealed multiple plasmid bands arranged in many different patterns, but multiple antibiotic resistances were shown to be commonly mediated by single transferable plasmids. Faecally-contaminated freshwater streams in Hong Kong may be reservoirs of antibiotic resistance plasmids for clinically-important bacteria.

Animals↗
PubMed1985

Expression of T4 early genes 62, 44, 45 and 46 in the lambda-T4 recombinant phage lambda 806-17.

A lambda-T4 recombinant phage, lambda 806-17, which carries the T4 early genes 62, 44, 45 and 46, was studied inside a homoimmune lysogen. Under such conditions, gene expression from the lambda promoters is represented. Results showed extensive expression of gene 46, and significant expression of genes 62 and 45. The expression of these early T4 genes is presumed to depend on T4 promoters included in the cloned fragment. A new promoter proximal to gene 46 is implicated. The results also indicate that the extent of gene expression, in terms of complementation, increases with the time allowed for expression.

Escherichia coli↗
PubMed1984

An improvement on labelled messenger RNA preparation for DNA-RNA hybridisation.

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled lambda cI47 late m-RNA is prepared and hybridised to the r-strain lambda DNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3%. This low background m-RNA preparation is important especially in dealing with minor class m-RNA.

Bacteriophage lambda↗
PubMed1983

Effect of multiplicity of infection on transcription in Escherichia coli cells infected by bacteriophage lambda.

The effect of multiplicity of infection (m.o.i.) on transcription was studied by infecting Escherichia coli with bacteriophage lambda cI47, lambda cI47O29P3 and lambda cI857cro27P3. DNA-RNA hybridization with lambda cI47 l-strand DNA, phi 80imm lambda r-strand DNA, and lambda imm80 r-strand DNA were used to measure mRNA transcription from the l-strand, the r-strand of the early x-P-Q region and the late A-J-b2 region of lambda bacteriophage respectively. In lambda cI47cro+O-P--infected cells, transcription from the l-strand, r-strand of the early x-P-Q region and the late A-J-b2 region all decreased with increasing m.o.i. The response in the x-P-Q region was less marked than in other regions, but the pattern looked similar to that described above. When phage DNA replication was permitted, as in the case of lambda cI47, the response was similar to that observed in lambda cI47cro+O-P--infected cells, but the level of transcription was increased two- or threefold. In lambda cI857cro-P--infected cells, the leftward transcription and the rightward transcription from the early x-P-Q region and the late A-J-b2 region all increased with increasing m.o.i., but the extent of change was less drastic than with lambda cro+. This result demonstrated clearly that the decrease in transcription from various regions at increasing m.o.i. of lambda cro+ was due to the inhibitory action of the cro gene product. The results obtained with cro- strongly support the view that gene dosage is a significant controlling factor for the extent of gene expression.

Bacteriophage lambda↗
PubMed1982

Gene dosage as a regulatory factor for gene expression. I. In lambda plac5-infected cells.

To study the effect of gene dosage on gene expression, lambda plac5cI857O29P3, a replication defective lambda phage carrying part of the lac operon (containing the lac promotor, operator and z gene) in the b2 region was studied in Escherichia coli strain JC6256 where the lac operon is deleted and at a temperature where the lambda repressor is inactive. In measuring the synthesis of beta-galactosidase, it was possible to separate the effects of the lac promoter from those of the phage promoter. When the synthesis of beta-galactosidase was initiated from the inserted lac promoter in JC6256(lambda +) in the presence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were directly proportional to the multiplicity of infection (gene dosage). Furthermore, beta-galactosidase synthesis was initiated about 5 min after infection, just as with isopropyl-beta-D-thiogalactoside (IPTG) induction. When the synthesis of beta-galactosidase was initiated from the phage promoter in JC6256 in the absence of additional cyclic AMP, the rate and level of beta-galactosidase synthesis were again linearly proportional to gene dosage. On the other hand, initiation of beta-galactosidase synthesis was delayed until 10 to 20 min after infection. These results suggest that: (i) in the absence of negative controlling factors, the extent of gene expression is proportional to gene dosage; (ii) varying the gene dosage can be used to regulate gene expression.

Bacteriophage lambda↗
PubMed1982

The time required for cro gene product to establish dominance in coliphage lambda lysogens.

To measure the length of heating required to convert a lambda lysogen in the immune (im+) phase into the anti-immune (im-) phase, rex gene activity was used as an indicator. It was observed that 5 min heating at 41 degrees C did not shift any lysogenic cells of 594 (lambda N-c1857O-) from the im+ phase into the im- phase, and it took 17 min heating at 41 degrees C followed by long hours of culture at 30 degrees C to shift half of the lysogenic cells into the im- phase. Such a length of heating is too long to be accounted for by blocking of the expression of lambda repressor by cro gene product. The result is more consistent with accumulation of a certain level of cro gene product during heating so that the synthesis of the repressor is blocked even after a return to low temperature.

Bacteriophage lambda↗
PubMed1982

A calorimetric study of the thermal transitions of Halobacterium cutirubrum.

The thermal transitions of Halobacterium cutirubrum have been examined by differential scanning calorimetry. Two distinct peaks corresponding to the denaturation of two major protein components were observed in the heating curves. One of the peaks has been assigned to the denaturation of the envelope glycoprotein. The variations of the denaturation temperatures with the addition of glucose, glycerol, NaNO3, and NaSCN are consistent with the previous proposal that hydrophobic interactions are essential in stabilizing the glycoprotein.

Calorimetry, Differential Scanning↗
PubMed1981

Purification of the gene 0.3 protein of bacteriophage T7, an inhibitor of the DNA restriction system of Escherichia coli.

The gene 0.3 protein of bacteriophage T7 prevents the DNA restriction system of EScherichia coli from interfering with T7 infection. A mutant strain of T7 that greatly overproduces the 0.3 protein has been constructed and used for purification of this protein. The 0.3 protein ws found to be extremely acidic and can be separated from virtually all other proteins of the infected cell by chromatography on DEAE-cellulose. Residual contaminating proteins and nucleic acids can be removed by gel filtration, but an even simpler final purification is possible, because under appropriate conditions the 0.3 protein is soluble in high concentrations of ethanol. Thus, a simple, essentially two-step purification can produce about 50 mg of pure 0.3 protein from 30 liters of culture. The purified protein appears to be a dimer of identical subunits. AS expected from its known function during infection, the purified 0.3 protein inhibits the nuclease and ATPase activities of partially purified Eco B, the DNA restriction enzyme of E. coli B, but it does not interfere with several different type II endonucleases tested. The inhibition of Eco B appears to require stoichiometric rather than catalytic amounts of 0.3 protein.

Adenosine Triphosphatases↗
PubMed1981

Amino acid sequence of the gene 0.3 protein of bacteriophage T7 and nucleotide sequence of its mRNA.

The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.

Amino Acid Sequence↗
PubMed1980

The phage promoter responsible for the expression of the inserted beta-galactosidase gene in bacteriophage lambda plac5.

The lac transducing phage, lambda plac5, carries a segment of the E. coli lac operon on the left side of the b2 region of the lambda phage. In the absence of additional cyclic AMP, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. This phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the cI and N gene products; however, the repressive action of the cro gene product at high multiplicity of infection is not observed although some turn off at very late time is detected. To pin down this phage promoter, results described in this communication and those described elsewhere can rule out the promoter PI, PR, P'R, and the promoter PL also looks rather unlikely. No firm identification of this phage promoter has been made, but the promoter(s) in the b2 region (the b2 promoter) is proposed. The phage promoter responsible for beta-galacrosidase synthesis is shown to be a weak promoter, requires the Q gene product or one (or more) of the late gene products for activation, and the time of expression is very late.

Bacteriophage lambda↗