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J Zieg

Publications and source records attributed to J Zieg.

13 recordsLinked to original sources
PubMed1989

The right end of MudI(Ap,lac).

Stable derivatives of the bacteriophage MudI-(Ap,lac) were used to generate operon fusions in S. typhimurium which exhibit a sectoring phenotype with respect to lacZ expression. The Lac- to Lac+ conversion was shown to be the result of small deletions involving the right end of the Mud/I element. DNA sequence analysis of several different fusions revealed that this end of MudI(Ap,lac) contains an assymetric inverted repeat of the attR site found in the wild-type Mu phage. A model is presented which explains how such a structure was formed in the construction of MudI(Ap,lac). In addition, this model explains the observed deletion formation and the Lac- to Lac+ conversion in the sectoring fusions.

Bacteriophage mu↗
PubMed1983

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.

Animals↗
PubMed1983

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

Animals↗
PubMed1980

Phase variation: evolution of a controlling element.

Phase variation in bacteria is regulated by homologous recombination at a specific DNA site. This recombinational event causes the inversion of a 970-base-pair DNA sequence that includes the promoter necessary for transcription of a flagellar gene. The invertible segment is flanked by two sites that are necessary for the inversion and contains a gene (hin) whose product mediates the inversion event. The hin gene shows extensive homology with the TnpR gene carried on the Tn3 transposon. It is also homologous with the gin gene carried on bacteriophage mu. These relationships suggest that the phase variation system may have evolved by the association of a transposon with a resident gene and the subsequent specialization of these elements to regulate flagellar antigen expression.

Amino Acid Sequence↗
PubMed1980

Analysis of the nucleotide sequence of an invertible controlling element.

The nucleotide sequence of the inversion region responsible for flagellar phase variation in Salmonella was determined. The inversion region is 995 base pairs (bp) in length and is bounded by a 14-bp inverted repeat sequence. A homologous recombination event between the 14-bp inverted repeat sequences would result in the inversion of the DNA segment between them. Sequence homologies with other systems suggest that the 14-bp inverted repeat sequences may have some general significance as sites for specific recombinational events. The gene which specifies H2 flagellin synthesis begins 16 bp outsie the inversion region. Within the inversion region, an open translational frame exists which could encode a low molecualr weight polypeptide (190 amino acids).

Amino Acid Sequence↗
PubMed1979

Phase variation in Salmonella: genetic analysis of a recombinational switch.

The alternative expression of Salmonella genes H1 and H2, which specify different flagellar antigens, results in the oscillation of phenotype known as phase variation. This alternation is controlled by the inversion of an 800-base-pair sequence of DNA adjacent to, or including part of, the H2 gene. The invertable region was presumed to regulate the function of a promoter and to include specific sites at which a recombinational event, resulting in the inversion, could occur. Here we report genetic manipulations of hybrid lambda phage carrying the H2 gene that were used to define the H2 promoter region and the recombinational sites. The H2 gene fragment was inserted on a hybrid lambda phage next to the cheW gene, which lacked a promoter element. In the resulting fusion, cheW gene activity was restored, the expression of the H2 and cheW genes was controlled coordinately by the inversion, and the polarity of transcription and location of the H2 gene could be determined. Evidence from this type of gene fusion suggested that the H2 gene promoter is included in the inversion region. Hybrid H2 phage were constructed that contained substitutions for regions of the H2 gene. In contrast to hybrid lambda containing the H2 gene, which alternate between "on" and "off" states, several substituted lambdaH2 were fixed in the "on" state. A site necessary for the recombinational event must have been removed in these fixed lambdaH2.

Bacterial Proteins↗
PubMed1979

Flagellar-phase variation: isolation of the rh1 gene.

In Salmonella, expression of flagellar antigen alternates between two serotypes (phases) encoded by two genes, H1 and H2. The mechanism which controls the alternative expression of the H1 and H2 genes was examined by cloning these genes and the genetic elements which control their activity on hybrid vehicles in Escherichia coli. H2 gene activity was shown to be controlled by a recombinational switch located adjacent to the H2 gene. Activity of the H1 gene is thought to be repressed, when the H2 gene is expressed, by the product of another gene, rh1 (repressor of H1), which is controlled coordinately with the H2 gene. In this report, we describe the construction of hybrid lambda vehicles which contain, in addition to the H2 gene, a genetic activity corresponding to rh1. Variation of flagellar antigens analogous to that observed in Salmonella was observed when E. Coli strains were transduced with the hybrid lambda. By using the lambdaH2rh1 hybrid to program protein synthesis in UV-irradiated cells, the synthesis of a polypeptide was correlated with rh1 gene product activity. We conclude that the H2 region consists of two cotranscribed genes, H2 and rh1. The expression of both gene products is regulated by the same recombinational event.

Bacterial Proteins↗
PubMed1978

Regulation of gene expression by site-specific inversion.

A site-specific inversion event is responsible for phase transition in Salmonella, as indicated by heteroduplex analysis of recombinant molecules carrying the gene coding for H2 flagellin in Salmonella. The inversion region corresponds to approximately 800 base pairs in length, and the inversion process does not appear to be dependent upon the E. coli RecA recombination pathway. Specific deletion derivatives of the cloned fragments no longer produce H2-specific flagella, effectively mapping the H2 gene within about 300 bp of the inversion region. Recombinant products of the hybrid molecules arose spontaneously, and they were used in the mapping of restriction sites within the inversion region. The restriction maps further demonstrate the extent and nature of the inversion.

Bacterial Proteins↗
PubMed1978

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products.

DNA Repair↗
PubMed1977

Recombinational switch for gene expression.

Flagellar antigens are specified by two genes, H1 and H2. The expression of these genes is regulated such that only one gene activity, or phase, is expressed at a given time. Molecular cloning techniques were used to isolate the segments of Salmonella DNA which contain these genetic loci. Heteroduplex analyses revealed an anomaly in the cloned fragment, that is, and apparent inversion, which was shown to be adjacent to the H2 gene. A correlation was demonstrated between the phase state of the H2 gene and the sequence of the adjacent segment. We propose that an inversion of this region is the phase-determining event in flagellar gene expression in Salmonella.

Antigens, Bacterial↗
PubMed1977

Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant.

A temperature-sensitive mutant of Chinese hamster cells is described which has two interesting properties: (1) it is a cell cycle mutant and (2) glycoprotein synthesis appears to be affected at the at the non-permissive temerature (40degreesC). Synchronized cells shifed to 40degreesC in the beginning of their G1 phase do not incorporate [3H]-thymidine into DNA during the expected S-phase, but once DNA synthesis has been initiated ( approximately 10 hours after termination of serum starvation) a shift to 40 degrees C no longer leads to an arrest of DNA synthesis. Flow microfluorimetric analysis of DNA content/cell supports this conclusion and indicates that a majority of cells become arrested in the G1 phase of the cell cycle when a non-synchronized population of cells is transferred to 40degreesC. Apparently at all times in the cell cycle there is a drastic reduction if incorporation of labeled sugars (particularly fucose) into glycoproteins. The uptake of fucose and its conversion to GDP-fucose appears to be normal at 40degreesC. Chromatographic analysis indicates that all classes of glycoproteins are affected, and we do not find any evidence for partially completed oligosaccharides at 40 degrees C. Overall protein synthesis is not reduced at he nonpermissive temperature during the time interval under consideration and the number of polysomes attached to membranes (RER) is also normal at 40degreesC. This suggests that the defect is at an early step in the synthesis or regulation of synthesis of glycoproteins. The mutation is a recessive mutation in hybrid cells and mutagen induced revertants can be obtained which grow normally at 40degreesC and in which glycoprotein synthesis at 40 degrees C is restored to normal, wild type levels.

Animals↗
PubMed1977

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12.

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.

Conjugation, Genetic↗