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J A Hewitt

Publications and source records attributed to J A Hewitt.

At least 19 recordsLinked to original sources
PubMed2002

Structure and characterization of the mouse UT-A gene (Slc14a2).

The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A (Slc14a2) and UT-B (Slc14a1). By genomic library screening and P1 artificial chromosome "shotgun" sequencing, we have determined the structure of the mouse UT-A gene. The gene is >300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5'-region of the gene is the UT-Aalpha promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-Abeta, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 microM dibutryl cAMP, 25 microM forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-Aalpha promoter construct 6.2-fold [from 0.026 +/- 0.003 to 0.160 +/- 0.004, relative light units (RLU)/microg protein] and a 2.4-kb UT-Abeta promoter construct 9.5-fold (from 0.020 +/- 0.002 to 0.190 +/- 0.043 RLU/microg protein) above that in untreated controls. Interestingly, only the UT-Abeta promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kg H(2)O with NaCl caused a significant increase (from 0.060 +/- 0.004 to 0.095 +/- 0.010 RLU/microg protein) in UT-Aalpha promoter activity but had no effect on the UT-Abeta promoter. A tonicity-responsive enhancer was identified in UT-Aalpha and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.

Animals↗
PubMed2001

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary.

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.

Animals↗
PubMed2000

Lck domains differentially contribute to pre-T cell receptor (TCR)- and TCR-alpha/beta-regulated developmental transitions.

Maturational changes at the CD4(-)CD8(-) double negative (DN) to CD4(+)CD8(+) double positive (DP) transition are dependent on signals generated via the pre-T cell receptor (TCR) and the nonreceptor protein tyrosine kinase p56(lck) (Lck). How Lck activities are stimulated or relayed after pre-TCR formation remains obscure. Our structure-function mapping of Lck thymopoietic properties reveals that the noncatalytic domains of Lck are specialized to signal efficient cellular expansion at DN to DP transition. Moreover, although substitution of the Lck catalytic domain with FynT sequences minimally impacts DP development, single positive thymocytes are most efficiently produced in the presence of kinases containing both the NH(2)-terminal and catalytic regions of Lck. These findings demonstrate that the Lck structure is uniquely adapted to mediate signals at both major transitions in thymopoiesis.

Animals↗
PubMed1998

The role of 2'-5' oligoadenylate-activated ribonuclease L in apoptosis.

Apoptosis of viral infected cells appears to be one defense strategy to limit viral infection. Interferon can also confer viral resistance by the induction of the 2-5A system comprised of 2'-5' oligoadenylate synthetase (OAS), and RNase L. Since rRNA is degraded upon activation of RNase L and during apoptosis and since both of these processes serve antiviral functions, we examined the role RNase L may play in cell death. Inhibition of RNase L activity, by transfection with a dominant negative mutant, blocked staurosporine-induced apoptosis of NIH3T3 cells and SV40-transformed BALB/c cells. In addition, K562 cell lines expressing inactive RNase L were more resistant to apoptosis induced by decreased glutathione levels. Hydrogen peroxide-induced death of NIH3T3 cells did not occur by apoptosis and was not dependent upon active RNAse L. Apoptosis regulatory proteins of the Bcl-2 family did not exhibit altered expression levels in the absence of RNase L activity. RNase L is required for certain pathways of cell death and may help mediate viral-induced apoptosis.

3T3 Cells↗
PubMed1996

Tissue-specific regulation of the WT1 locus.

The 11p13 Wilms' tumor locus consists of two coordinately regulated transcripts, WT1 and WIT-1. These genes are highly expressed in the developing urogenital system, beginning with the urogenital ridge at day 10.5, the metanephric blastema at day 11.5, and during glomerular formation at day 13.5, becoming ultimately restricted to the podocytes. Stromal cells of the gonad also show abundant expression. WT1 is expressed at lower levels in spleen, uterus, mesothelial linings of organs in the abdominal and thoracic cavities, and the ependymal layer of the ventral aspect of the spinal cord. WIT-1 mRNA is about 10-fold less abundant than WT1, but appears to be expressed in the same tissue-restricted manner. Expression of the WT1 protein is required for kidney development, although its physiological function remains to be determined. The function of WIT-1 is similarly unknown but one intriguing possibility is that it is an antisense regulator of WT1. An understanding of events controlling spatial and temporal regulation of these genes will greatly improve our ability to study the role of WT1 and WIT-1 in urogenital development. We have found that while chimeric reporter constructs containing 0.6-2.5 kb of the 5' region of the WT1 gene direct transcription in many different cell lines, we were unable to detect expression in 13.5-day mouse embryos. However, a cosmid containing about 42 kb encompassing this region was able to direct the expression of abundant levels of mRNA from the appropriate transcription initiation sites in both stable transfectants of mouse Leydig cells (TM3) or in transgenic embryos. We are currently localizing the DNA elements required for this expression.

Animals↗
PubMed1994

Antisense transcripts and protein binding motifs within the Wilms tumour (WT1) locus.

Transcription of the WT1 locus is restricted, both temporally and spatially, to a subset of epithelial cells in mammalian kidneys and gonads. WT1, one of the two divergent transcripts mapping to this locus encodes a zinc finger protein that is likely a transcriptional regulator. The other transcript, WIT1, encodes a product of unknown function that is subject to alternate splicing in the region immediately 5' of the WT1 gene. Analysis of the 5' end of this locus further revealed the presence of multiple transcriptional start sites for both genes, such that some of the WIT1 transcripts are encoded by the antisense strand of the first exon of WT1. The genomic region surrounding the transcriptional start sites appears to constitute part of a bi-directional promoter based on the ability of a DNA fragment derived from this region to direct expression of a chimeric CAT gene construct in transient transfection assays. Discrete sequences within the region are capable of interaction in vitro with nuclear extracts derived from a variety of rat and mouse tissues. Interestingly, recombinant WT1, representing the product of zinc finger region of the most abundant of the four alternatively spliced transcripts, is also capable of binding to sequences within this region.

Animals↗
PubMed1983

Chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly.

The major (gene VIII) coat protein of bacteriophage fd was radiolabelled by treating the virus with methyl[3H]acetimidate without causing any loss of infectivity. Complete amidination of lysine-8 in the amino acid sequence of the protein was achieved but little or no modification of the lysine residues near the C terminus was observed. This supports the assumption that the coat protein is oriented in the viral filament with its N terminus on the outside and its C-terminal region abutting the DNA. Escherichia coli was co-infected with radiolabelled bacteriophage and with unlabelled miniphage, a shorter defective form of phage fd. Radiolabel was detected in the progeny miniphage, proving that individual coat protein subunits can be recycled and assembled onto progeny miniphage DNA. About 35% of the coat protein subunits of phage particles infecting E. coli were recycled in 1 h. These facts support a model of the assembly and disassembly of the virion at the bacterial membrane in which the end of the particle containing the minor adsorption (gene III) protein, which is presumably the first to disassemble during infection, is the last to assemble during morphogenesis.

Amino Acids↗
PubMed1978

Controlled proteolytic digestion of the M-protein of Sendai virus: the isolation of a fragment of 30000 molecular weight.

Proteolytic digestion of the M-protein of Sendai virus produces a product with a mol. wt. approximately 5000 less than that of the intact protein. In the case of digestion with chymotrypsin this cleavage is quite specific and the cleaved protein can be isolated. The smaller fragment appears to be physically removed from the larger (30000 mol. wt.) fragment, rather than remaining in non-covalent association with it. The cleavage is likely to be near the N-terminus of the protein. At the present time there is no indication of the biological function of this fragment.

Amino Acids↗
PubMed1977

A morphological study of the M-protein of Sendai virus.

A purification scheme is described for the M-protein of Sendai virus and an electron microscope study of the isolated protein is presented. The protein exists as subunits of 6 nm in diam., which possess a central hole; the subunits may be dimers of the polypeptide. They are able to form filamentous aggregates which wind around one another to form a helical structure. It is suggested that these filaments may be the form adopted by the protein in the virus, the filaments lying parallel to one another just beneath the virus membrane to form a shell, but that the helical form is likely to be a property only of the isolated protein.

Microscopy, Electron↗
PubMed1975

Miniphage-a class of satellite phage to M13.

Satellite or defective bacteriophage particles can appear in extensively recycled stocks of coliphage M13. These particles, herein known as miniphage, replicate using the wild type bacteriophage as a helper. Their physical properties (u.v. spectra, sedimentation of DNA and bacterophage, electrophoretic moblitiy) are described and a method for the isolation of specific satellite bacteriophage is presented.

Amino Acids↗