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Biomedical subjects

M F Wilkinson

Publications and source records attributed to M F Wilkinson.

At least 19 recordsLinked to original sources
PubMed2001

An active immunization approach to generate protective catalytic antibodies.

We report that mice immunized with a phosphate immunogen produced polyclonal catalytic antibodies (PCAbs) that catalysed the hydrolysis of carbaryl, a widely used broad-spectrum carbamate insecticide that exerts toxic effects in animals and humans. The reaction catalysed by the PCAbs (IgGs) obeyed Michaelis-Menten kinetics in vitro with the following values at pH 8.0 and 25 degrees C: K(m) approximately 8.0 microM, k(cat)=4.8x10(-3)-5.8x10(-1), k(cat)/k(non-cat)=5.6x10(1)-6.8x10(3) (where k(non-cat) is the rate constant of the reaction in the absence of added catalyst). The PCAbs were also active in whole sera under physiological conditions in vitro. The PCAbs induced in vivo were also active in vivo, as immunization with the phosphate immunogen decreased the mouse blood concentration of carbaryl. To our knowledge, this is the first report demonstrating that active immunization generates antibodies possessing therapeutic catalytic function in vivo. We propose that active immunization schemes that induce enzymically active antibodies may provide a highly specific therapeutic approach for degrading toxic substances.

Animals↗
PubMed2001

Localization and stability of introns spliced from the Pem homeobox gene.

RNA splicing generates two products in equal molar amounts, mature mRNAs and spliced introns. Although the mechanism of RNA splicing and the fate of the spliced mRNA products have been well studied, very little is known about the fate and stability of most spliced introns. Research in this area has been hindered by the widely held view that most vertebrate introns are too unstable to be detectable. Here, we report that we are able to detect all three spliced introns from the coding region of the Pem homeobox gene. By using a tetracycline (tet)-regulated promoter, we found that the half-lives of these Pem introns ranged from 9 to 29 min, comparable with those of short lived mRNAs such as those encoding c-fos and c-myc. The half-lives of the Pem introns correlated with both their length and 5' to 3' orientation in the Pem gene. Subcellular fractionation analysis revealed that spliced Pem introns and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, and DNase-sensitive fractions within the nucleus. Surprisingly, we found that all three of the spliced Pem introns were also in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. This indicates either that spliced Pem introns are uniquely exported to the cytoplasm for degradation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled.

Animals↗
PubMed2001

Multifunctional regulatory proteins that control gene expression in both the nucleus and the cytoplasm.

The multistep pathway of eukaryotic gene expression involves a series of highly regulated events in the nucleus and cytoplasm. In the nucleus, genes are transcribed into pre-messenger RNAs which undergo a series of nuclear processing steps. Mature mRNAs are then transported to the cytoplasm, where they are translated into protein and degraded at a rate dictated by transcript- and cell-type-specific cues. Until recently, these individual nuclear and cytoplasmic events were thought to be primarily regulated by different RNA- and DNA-binding proteins that are localized either only in the nucleus or only the cytoplasm. Here, we describe multifunctional proteins that control both nuclear and cytoplasmic steps of gene expression. One such class of multifunctional proteins (e.g., Bicoid and Y-box proteins) regulates both transcription and translation whereas another class (e.g., Sex-lethal) regulates both nuclear RNA processing and translation. Other events controlled by multifunctional proteins include assembly of spliceosome components, spliceosome recycling, RNA editing, cytoplasmic mRNA localization, and cytoplasmic RNA stability. The existence of multifunctional proteins may explain the paradoxical involvement of the nucleus in an RNA surveillance pathway (nonsense-mediated decay) that detects cytoplasmic signals (premature termination codons). We speculate that shuttling multifunctional proteins serve to efficiently link RNA metabolism in the cytoplasmic and nuclear compartments.

Animals↗
PubMed2001

Precursor RNAs harboring nonsense codons accumulate near the site of transcription.

Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.

Animals↗
PubMed2001

Irradiation selectively inhibits expression from the androgen-dependent Pem homeobox gene promoter in sertoli cells.

How radiation blocks spermatogenesis in certain strains of rats, such as LBNF(1), is not known. Because the block depends on androgen, we propose that androgen affects Sertoli cell function in irradiated LBNF(1) rats, resulting in the failure of spermatogonial differentiation. To begin to identify genes that may participate in this irradiation-induced blockade of spermatogenesis, we investigated the expression of several Sertoli genes in response to irradiation. The expression of the PEM: homeobox gene from its androgen-dependent Sertoli-specific proximal promoter (Pp) was dramatically reduced more than 100-fold in response to irradiation. In contrast, most other genes and gene products reported to be localized to the Sertoli cell, including FSH receptor (FSHR), androgen receptor (AR), SGP1, and the transcription factor CREB, did not exhibit significant changes in expression, whereas transferrin messenger RNA (mRNA) expression dramatically increased in response to irradiation. Irradiation also decreased Pp-driven PEM: mRNA levels in mouse testes (approximately 10-fold), although higher doses of irradiation than in rats were required to inhibit PEM: gene expression in testes of mice, consistent with their greater radioresistance. The decrease in Pem gene expression in mouse testis was also selective, as the expression of CREB, GATA-1, and SGP1 were little affected by irradiation. We conclude that the dramatic irradiation-triggered reduction of Pem expression in Sertoli cells is a conserved response that may be a marker for functional changes in response to irradiation.

Androgens↗
PubMed2000

Rapid induction of nuclear transcripts and inhibition of intron decay in response to the polymerase II inhibitor DRB.

The transcriptional inhibitor 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) is an adenosine analog that has been shown to cause premature transcriptional termination and thus has been a useful tool to identify factors important for transcriptional elongation. Here, we establish an efficient system for studying DRB-sensitive steps of transcriptional elongation. In addition, we establish two novel effects of DRB not previously reported: intron stabilization and the induction of long transcripts by a mechanism other than premature termination. We found that DRB had a biphasic effect on T-cell receptor-beta (TCRbeta) transcripts driven by a tetracycline (tet)-responsive promoter in transfected HeLa cells. In the first phase, DRB caused a rapid decrease (within five minutes) of pre-mRNA and its spliced intron (IVS1(Cbeta1)), consistent with the known ability of DRB to inhibit transcription. In the second phase (which began ten minutes to two hours after treatment, depending on the dose), DRB dramatically increased the levels of IVS1(Cbeta1)-containing transcripts by a mechanism requiring de novo RNA synthesis. DRB induced the appearance of short 0.4 to 0.8 kb TCRbeta transcripts in vivo, indicating DRB enhances premature transcriptional termination. A approximately 475 nt prematurely terminated transcript (PT) was characterized that terminated at an internal poly(A) tract in the intron IVS1(Cbeta1). We identified three other effects of DRB. First, we observed that DRB induced the appearance of heterodisperse TCRbeta transcripts that were too long ( approximately 1 kb to >8 kb) to result from the type of premature termination events previously described. Their production was not promoter-specific, as we found that long transcripts were induced by DRB from both the tet-responsive and beta-actin promoters. Second, DRB upregulated full-length normal-sized c-myc mRNA, which provided further evidence that DRB has effects besides regulation of premature termination. Third, DRB stabilized lariat forms of the intron IVS1(Cbeta1), indicating that DRB exerts post-transcriptional actions. We propose that our model system will be useful for elucidating the factors that regulate RNA decay and transcriptional elongation in vivo.

Cell Nucleus↗
PubMed1999

The stability and fate of a spliced intron from vertebrate cells.

Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1Cbeta1, the first intron from the constant region of the mouse T-cell receptor-beta, (TCR-beta) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1Cbeta1 and its pre-mRNA had half-lives of 6.0+/-1.4 min and 3.7+/-1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1Cbeta1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1Cbeta1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1Cbeta1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1Cbeta1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1Cbeta1 also appeared to be selectively exported to the cytoplasm, whereas TCR-beta pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.

Animals↗
PubMed1998

Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo.

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3' processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523-527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

Amino Acid Sequence↗
PubMed1998

Distal V beta promoters transcribe novel T-cell receptor-beta transcripts in early development.

The transcriptional activation of germline T-cell receptor (TCR) and immunoglobulin (Ig) genes has been proposed to promote the rearrangement of these genes. Here we report the identification of distal TCR promoters (PDs), located upstream of the previously characterized promoters in the mouse V beta 5.1 and V beta 8.1 gene segments, that are active in germline TCR genes in fetal thymus and liver in vivo. We also identified an immature T-cell clone, SL12.4, that expresses both endogenous and transfected PDs in a regulated manner in vitro. We propose that the transcription of these distal promoters in germline TCR genes may be important for inducing TCR gene rearrangements during T-cell development. Northern blot, RNase protection and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that PDs are also transcribed from fully rearranged TCR genes in adult thymus, lymph node, and spleen. Although the functional significance of this expression is not known, our sequence analysis of the 5' leader in PD-derived V beta 5.1 and V beta 8.1 transcripts revealed the presence of several open reading frames (ORFs) that may encode novel polypeptides or regulate the efficiency of TCR beta translation.

Animals↗
PubMed1997

The rapidly evolving Pem homeobox gene and Agtr2, Ant2, and Lamp2 are closely linked in the proximal region of the mouse X chromosome.

The Pem gene encodes a homeodomain-containing protein expressed in reproductive tissue that may function as a transcription factor regulating spermatogenesis and sperm maturation. We have mapped the Pem gene to the proximal end of the mouse X chromosome, placing it within the Hprt region. Based on the mapping of Pem and other loci in three separate Mus musculus x Mus spretus backcross panels, we established the order of markers within this segment of the Hprt region as: Agtr2-Pem-Ant2-DXMit50-Lamp2-DXMit49. In contrast to some other regions of the X chromosome, which have been rearranged during the evolution of mammals, we show that the order of gene loci within this Hprt region is conserved in mice and human. The finding that the mouse Ant2 and Pem loci are tightly linked suggests that human ANT2 may be useful as a marker for isolating the human PEM gene, which has been impervious to cloning by conventional hybridization methods because of its rapid evolution.

Animals↗
PubMed1997

T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism.

Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-beta gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-beta exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.

Animals↗
PubMed1997

A processed homeobox gene expressed in a stage-, tissue- and region-specific manner in epididymis.

Processed genes are generated by reverse transcription of mRNA and integration at a novel site in the genome and are typically transcriptionally silent. Here, we report that a processed gene on rat chromosome 4 that is highly related to the X chromosome-encoded rat Pem (r.Pem) homeobox gene is transcriptionally active. This processed gene, r.Pem2, is expressed at high levels in epididymis but not in any other tissues that express the r.Pem gene, including testis. Although r.Pem2, is expressed at high levels in epididymis but not in any other tissues that express the r.Pem gene, including testis. Although r.Pem2 lacks all the introns present in the r.Pem gene, it contains splice donor and acceptor sequences within the coding region, permitting it to be spliced and to potentially encode a 57 amino acid polypeptide. r.Pem2 transcripts accumulate in the caput and cauda regions of the epididymis but not in the initial segment. The r.Pem2 gene exhibits different regulation from the r.Pem gene; its expression is induced later during prepubertal development (between days 23 and 30 post partum) and independently of the presence of testosterone. Although the functional significance of r.Pem2 is unknown, its developmental regulation and its apparent acquisition of splicing sites during evolution are unique.

Amino Acid Sequence↗
PubMed1997

Rapid evolution of a homeodomain: evidence for positive selection.

One often-noted feature of homeobox genes is the conservation of the homeodomain among orthologous genes from distantly related species. This sequence conservation is presumed to reflect functional conservation, which indeed has been demonstrated in several cases. We analyzed the evolution of an orphan homeobox gene, Pem, which is expressed preferentially in male and female reproductive tissue. Sequence analysis of 12 species of mice and rats indicated that the Pem gene has evolved at a remarkably high rate. The most rapidly evolving region of the Pem protein is the amino portion of the homeodomain, including the flexible N-terminal arm, helices I and II, and the linker regions between the helices. In contrast, the third helix, which is known to mediate base-specific DNA contacts in other homeodomains, is conserved in the Pem protein. Analysis of the ratio of nonsynonymous and synonymous codon substitution rates within the Pem homeodomain suggested that its divergence was driven by adaptive selection. The rate of nonsynonymous substitutions in Pem was higher than that of the sex-determination gene Sry, which also appears to have undergone directional selection over a short evolutionary period. Despite the rapid evolution of the Pem gene, we detected no Pem polymorphisms and observed no variation in the homeobox sequence among closely related Mus species. This suggests that purifying episodes followed phases in which selection pressure drove the rapid divergence of this locus. We propose that transcription factors that function in reproductive events can be subject to rapid adaptive selection.

Amino Acid Sequence↗