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C D Sigmund

Publications and source records attributed to C D Sigmund.

85 records · Page 5Linked to original sources
PubMed1990

Expression of murine renin genes during fetal development.

Fetuses were examined to produce a developmental profile of renin expression in the kidneys and adrenal glands in single renin gene and two renin gene strains of mice. Sites of renin expression were detected by in situ hybridization using an 35S-labeled antisense RNA probe complimentary to the renin cDNA. Accumulation of renin transcripts in the adrenal gland reached a maximum at 15.5 days post coitum for all strains examined, but declined to undetectable levels by birth in one gene strains, while in two gene strains, the levels of renin transcripts lessened and by birth became limited to the developing inner cortex. Kidney renin transcripts were first detected at 14.5 days post coitum in the newly developing arteries in fetuses of both genotypes of mice. As the renal arterial tree developed, renin mRNA containing cells were progressively localized to more distal blood vessels and finally to the specialized cells of the afferent arteriole (juxtaglomerular cells). These results were confirmed by examining the localization of immunoreactive T antigen in transgenic fetuses. These mice carried a transgene which placed the SV40 T antigen structural gene under control of renin regulatory elements. Expression of T antigen occurred at the same sites in the kidneys and adrenal glands as renin mRNA. Furthermore, in strains with two renin genes, primer extension analysis indicated transcripts from both genes were present in equal proportion in combined kidney and adrenal gland extracts of total RNA. These transcripts were full length in size. The transient localization of renin mRNA in cells of the fetal intrarenal arteries is consistent with the notion that renin may be a useful marker for the developing renal vasculature.

Adrenal Glands↗
PubMed1989

Expression of the DBA/2J Ren-2 gene in the adrenal gland of transgenic mice.

To characterize further the tissue-specific control of the mouse Ren-2 gene, and in particular its expression in the adrenal gland, we have introduced the DBA/2 Ren-2 gene into the genome of Ren-1c/Ren-1c mice. Here we report our observations on Ren-2 transgenic mice. Expression was found in the correct spectrum of tissues and included appropriate hormonal control in the submandibular gland. Quantitatively transcript levels varied both positively (adrenal gland and sex-accessory tissue) and negatively (submandibular gland and kidney) with respect to normal Ren-2 expression. In the DBA/2 inbred mouse strain expression in the female adrenal gland cycles during oestrus between the X-zone and the zona fasciculata. Transgene expression within the adrenal gland was restricted to the X-zone. Therefore this phenotype, which is characteristic of most two-renin-gene strains of mice, contrasts with that found in the strain DBA/2 from which the transgene was derived. This suggests that cell-specific expression in the DBA/2 adrenal gland is mediated in trans by at least one additional locus. We demonstrate that suitable genetic crosses of the transgenic mice can partially restore the cycling phenotype.

Adrenal Glands↗
PubMed1989

Allele-specific expression of the murine Ren-1 genes.

Inbred strains of mice carry either one of two alleles for the Ren-1 gene on chromosome 1; Ren-1c or Ren-1d. The Ren-1d allele is found in association with a tightly linked duplicated locus, Ren-2. The relative accumulations of renin transcripts derived from each locus were found to differ between mice with one or two renin loci in three extra renal tissues, the adrenal gland, testis, and sex accessory gland tissue. We find that the two Ren-1 alleles exhibit characteristic expression patterns in these tissues. Using genetic analyses and an allele-specific dideoxynucleotide primer extension assay we have further characterized the expression differences in these tissues. The results suggest that the allele-specific expression patterns observed for the Ren-1 genes are regulated by closely associated sequences in cis.

Adrenal Glands↗
PubMed1988

Nus A protein affects transcriptional pausing and termination in vitro by binding to different sites on the transcription complex.

We examined the in vitro concentration dependence of the effects of Nus A on transcription termination and pausing to determine if Nus A affects both pausing and termination in vitro by binding to a single site on the transcription complex. Nus A was shown to cause maximal increases of pausing at a concentration approximately equimolar to RNA polymerase. However, the effects of Nus A on termination require much higher Nus A concentrations than are required for pausing. It is therefore likely that the effects of Nus A on pausing and termination result from the binding of Nus A to different sites on the transcription complex. Since proteins that probably bind RNA nonspecifically were also shown to strongly reduce termination at a Rho-dependent terminator, Nus A may decrease Rho-dependent termination by binding nonspecifically to RNA. This proposal is consistent with most of the available data on the in vitro effects of Nus A and provides a mechanistic basis for previously unexplained details of Nus A caused decreases in Rho-dependent termination. We further speculate that most or all of the in vivo roles of Nus A may involve the enhancement of pausing.

Bacterial Proteins↗
PubMed1988

Effects of Escherichia coli Nus A protein on transcription termination in vitro are not increased or decreased by DNA sequences sufficient for antitermination in vivo.

The ability of Escherichia coli Nus A protein to recognize specific DNA or RNA sequences in vitro was tested by using transcription templates containing a variety of promoters, transcription terminators, and antitermination-conferring regions. We conclude that the effects of Nus A on termination are not qualitatively or quantitatively altered by sequences present in promoters, Rho-dependent terminators, or antitermination-conferring regions. Nus A was also shown to increase termination at the rrnC Rho-independent T1 terminator by a mechanism that is independent of the promoter or sequences involved in antitermination. Altogether, these observations argue against a direct Nus A-nucleic acid interaction affecting termination in vitro. Together with the results described in the accompanying paper [Sigmund, C. D., & Morgan, E. A. (1988) Biochemistry (preceding paper in this issue)], these results suggest that the effects of Nus A on termination in vitro may not be related to the in vivo functions of Nus A.

Bacterial Proteins↗
PubMed1988

Studies on the regulation of renin genes using transgenic mice.

Transgenic mice are being used as an assay to identify the DNA sequences that direct the tissue specificity and physiological regulation of murine renin gene expression. In initial studies, a DNA segment encoding the duplicated renin gene, Ren-2, has been injected into the pronuclei of fertilized eggs homozygous for the Ren-1c allele. The 24 kb XhoI fragment utilized consists of 5.3 kb of upstream flanking sequences and 9.5 kb of 3' flanking sequence in addition to the 10 kb segment encoding the 9 exons of the natural gene. Seventeen transgenic founder mice were identified of which at least 7 exhibited evidence of transgene expression. The 4 expressing lines that have been studied in detail integrated from 5 to 15 copies of the transgene but expression was not proportional to the number of copies integrated. Qualitatively, expression is detected in the correct spectrum of tissues, and submandibular gland expression of the transgene responds appropriately to known hormonal modulators. However, quantitatively, the levels of expression deviate considerably from normal.

Animals↗
PubMed1984

Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of Escherichia coli.

Recombinant DNA and classic genetic procedures were used to map a spectinomycin resistance mutation to a 121 base pair region of a 16S RNA gene and a macrolide-lincosamide-streptogramin type B resistance mutation to a 32 base pair region of a 23S RNA gene. DNA sequence analysis of these regions revealed that spectinomycin resistance results from a C/G to T/A transition at position 1192 of a 16S RNA gene. Resistance to macrolide, lincosamide and streptogramin type B antibiotics results from an A/T to T/A transversion at position 2058 of a 23S RNA gene. The alteration in 16S RNA is in a sequence that can participate in alternate base pairing arrangements that have been proposed to be involved in the translocation process. The alteration in 23S RNA identifies sequences important to peptidyl transfer.

Base Sequence↗
PubMed1983

Spectinomycin resistance due to a mutation in an rRNA operon of Escherichia coli.

A spectinomycin resistance mutation was isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Cell-free protein-synthesizing extracts made from cells containing the plasmid were partially resistant to spectinomycin. Although spectinomycin is an aminoglycoside antibiotic, the mutation did not confer resistance to any other aminoglycoside antibiotic tested.

Aminoglycosides↗
PubMed1982

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.

DNA, Recombinant↗
PubMedDate not supplied

Modifiable gene expression in mice: kidney-specific deletion of a target gene via the cre-loxP system.

With the advent of gene-targeting in mouse embryonic stem (ES) cells, the use of knockout mice to study the physiological effects of loss of gene function has become increasingly prevalent. However, there are several drawbacks with conventional gene-targeting approaches which may make phenotyping of the resultant mice difficult, if not, impossible. Conventional gene-targeting results in the loss of function of the targeted gene in all cells and tissues, which can be problematic for genes which are required developmentally, which exhibit a wide tissue-specific expression pattern, or are part of complex paracrine systems. As with mice that lack the angiotensinogen or endothelin-1 gene, loss of gene function may lead to a lethal phenotype which can be manifested during embryonic development, at birth or postnatally. These limitations could potentially be circumvented by using a system in which the loss of gene function is placed under spatial and/or temporal control. We will discuss how the cre-loxP recombinase system can be applied to delete a gene in a tissue- and developmentally regulated fashion.

Animals↗
PubMedDate not supplied

Kidney is the only source of human plasma renin in 45-kb human renin transgenic mice.

Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.

Animals↗