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The Wilms' tumor suppressor WT1: approaches to gene function.

Occurring with a frequency of 1 in 10,000 live births, Wilms' tumor is one of the most common solid tumors of children. The genetic basis of this tumor is highly complex and several loci have been shown to be associated with tumor formation. Thus far, however, WT1 is the only gene that has been isolated and proven to carry mutations within Wilms' tumors. During the last few years, a wealth of experiments has been carried out to address the function of WT1 as a tumor suppressor and developmental regulator. This review focuses on studies addressing WT1 function; new approaches to understand WT1 function in vivo and present transgenic data in which WT1 was driven ectopically using a CMV promoter are discussed. Our results suggest that ubiquitous expression of WT1 is not compatible with embryonic development.

Culture Techniques↗

Herpes simplex virus latency-associated transcript gene function.

A major area of interest in the study of herpes simplex virus type 1 (HSV-1) involves the persistence of the virus within a latent state in neuronal cells of infected humans. The latency-associated transcripts (LATs) are believed to play a key role during HSV-1 latency. This review will discuss the most recent findings on the involvement of the LAT region with apoptotic pathways and how this relates to other potential functions of the LATs.

Animals↗

The human S3a ribosomal protein: sequence, location and cell-free transcription of the functional gene.

The intron-containing gene encoding human ribosomal protein S3a (hRPS3a) was isolated by utilizing a PCR-based strategy to detect a gene-specific intron which was subsequently used as a probe for cloning of the entire gene. The hRPS3a gene is composed of six exons and five introns spanning 5013 bp. As described for other hRP-encoding genes, the promoter lacks a canonical TATA sequence and a defined CAAT box. Primer extension experiments, as well as cell-free transcription, revealed that a cytosine functions as the major transcription start point in a polypyrimidine region, but a guanosine at position -1 was also able to initiate transcription. Hybridization analysis of chromosomal DNA from a panel of human-rodent somatic cell hybrids revealed that hRPS3a is encoded by a single locus in the human genome, present on chromosome 4.

Amino Acid Sequence↗

Control of gene function in baceriophage T4. II. Synthes of messenger ribonucleiccid and proei after interrupting deoxyribonucleic acid replication and glucosylation.

Replication of T4 deoxyribonucleic acid (DNA) is known to be required for the onset of transcription of late T4 genes. Once late gene transcription has been initiated, DNA replication is no longer required for maintaining synthesis of late or early T4 messenger ribonucleic acid (mRNA). Late phage proteins (lysozyme and tail fibers) continue to be produced at constant rates after interrupting T4 DNA synthesis. The ability of the host cell to glucosylate the T4 progeny DNA has no demonstrable influence on the rates at which T4 mRNA and late proteins are synthesized after the interruption of DNA synthesis. To explain the requirement of T4 DNA replication for the onset of late gene transcription, we suggest that T4 DNA in a nascent state is mandatory for the initial late gene transcription, or perhaps for late gene transcription throughout the lytic cycle. T4 DNA in a nascent state could be segregated from the bulk of the replicating DNA, used only as template for RNA synthesis, and prevented from being modified by methylation, glucosylation, or maturation processes. The fact that no, or very little, nonglucosylated T4 DNA is extractable from T4LB3-infected CR63 after arresting DNA synthesis does not rule out this possibility.

Centrifugation, Density Gradient↗

DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity.

Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.

Adenine↗

RNA silencing as a tool for exploring gene function in ascomycete fungi.

We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.

Base Sequence↗

Gene function analysis in environmental isolates: the nif regulon of the strict iron oxidizing bacterium Leptospirillum ferrooxidans.

A random genomic library from an environmental isolate of the Gram-negative bacterium Leptospirillum ferrooxidans has been printed on a microarray. Gene expression analysis was carried out with total RNA extracted from L. ferrooxidans cultures in the presence or absence of ammonium as nitrogen source under aerobic conditions. Although practically nothing is known about the genome sequence of this bacterium, this approach allowed us the selection and sequencing of only those clones bearing genes that showed an altered expression pattern. By sequence comparison, we have identified most of the genes of nitrogen fixation regulon in L. ferrooxidans, like the nifHDKENX operon, encoding the structural components of Mo-Fe nitrogenase; nifSU-hesB-hscBA-fdx operon, for Fe-S cluster assembly; the amtB gene (ammonium transporter); modA (molybdenum ABC type transporter); some regulatory genes like ntrC, nifA (the specific activator of nif genes); or two glnB-like genes (encoding the PII regulatory protein). Our results show that shotgun DNA microarrays are very powerful tools to accomplish gene expression studies with environmental bacteria whose genome sequence is still unknown, avoiding the time and effort necessary for whole genome sequencing projects.

Bacteria↗

rpoS gene function is a disadvantage for Escherichia coli BJ4 during competitive colonization of the mouse large intestine.

The ability of Escherichia coli to survive stress during growth in different environments is, in large part, dependent on rpoS and the genes that comprise the rpoS regulon. E. coli BJ4 and an isogenic BJ4 rpoS mutant were used to examine the influence of the rpoS gene on E. coli colonization of the streptomycin-treated mouse large intestine. Colonization experiments in which the wild-type E. coli BJ4 and its rpoS mutant were fed individually as well as simultaneously to mice suggested that E. coli BJ4 does not face prolonged periods of nutrient starvation in the mouse large intestine and that the rpoS regulon is not expressed during long-term colonization after adaptation of the bacteria to the gut environment.

Animals↗

Functional gene analysis of individual response to challenge of SIVmac239 in M. mulatta PBMC culture.

It has previously been shown in macaques that individual animals exhibit varying responses to challenge with the same strain of SIV. We attempted to elucidate these differences using functional genomics and correlate them to biological response. Unfractionated PBMC from three rhesus macaques were isolated, activated, and infected with SIVmac239. Interestingly, one of the three animals used for these experiments exhibited a completely unique response to infection relative to the other two. After repeated attempts to infect the PBMC from this animal, little or no infectivity was seen across the time points considered, and corresponding to this apparent lack of infection, few genes were seen to be differentially expressed when compared to mock-infected cells. For the remaining two animals, gene expression analysis showed that while they exhibited responses for the same groups of pathways, these responses included differences specific to the individual animal at the gene level. In instances where the patterns of differential gene expression differed between these animals, the genes being differentially expressed were associated with the same categories of biological process, mainly immune response and cell signaling. At the pathway level, these animals again exhibited similar responses that could be predicted based on the experimental conditions. Even in these expected results, the degree of response and the specific genes being regulated differed greatly from animal to animal. The differences in gene expression on an individual level have the potential to be used as markers in identification of animals suitable for lentiviral infection experiments. Our results highlight the importance of individual variation in response to viral challenge.

Animals↗