Biomedical subjects

M S Bradshaw

Publications and source records attributed to M S Bradshaw.

9 recordsLinked to original sources

PubMed1996

A long-range regulatory element of Hoxc8 identified by using the pClasper vector.

Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene.

Animals↗

PubMed1995

A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.

The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.

Base Sequence↗

PubMed1995

Human HOXB cluster and the nerve growth factor receptor gene: comparison with an orthologous chromosomal domain in mouse.

The structural organization and nucleotide sequence similarity of mammalian Antennapedia-class homeobox genes support the view that the four homeobox clusters (HOXA, B, C, and D on human chromosomes 7, 17, 12, and 2, respectively) arose through a combination of gene duplication and divergence to form a cluster, followed by several cluster duplications. The duplication events that gave rise to the four clusters appear to have involved chromosomal domains extending well beyond the borders of the clusters in either direction. This evidence arises from the observation that many genes closely linked to the homeobox clusters on different chromosomes show sequence similarity. Here, we present a continuation of physical mapping studies to determine the extent and organization of the duplicated regions surrounding the four homeobox clusters in human. Southern blots prepared from pulsed-field gels of human DNA were probed with cloned segments of human HOXB genes and the nerve growth factor receptor (NGFR) gene on chromosome 17q21-q22. Restriction enzyme analysis revealed the close physical linkage of these genes within 100 kb. Two yeast artificial chromosomes (YACs), 220 and 380 kb in size, were isolated using oligonucleotide primers specific for NGFR. Both YACs contained the entire HOXB cluster. Restriction mapping of the clones indicated that the distance separating these loci could not be greater than 50 kb. This result confirms and extends previous information on the proximity of these genes as determined by genetic linkage analysis and closely parallels the orthologous loci in the mouse.

Animals↗

PubMed1994

Identification of the murine Hox-c12 and Hox-c13 homeoboxes on yeast artificial chromosomes.

The structure of the Antennapedia-class homeobox gene clusters is highly conserved in human and mouse. Of all the human homeobox genes known, only HOX-C12 and HOX-C13 have not also been identified in mouse. These two genes are located at the extreme 5' end of the human HOX-C cluster and are predicted to be located in a similar position on the murine Hox-c cluster. We have isolated two yeast artificial chromosomes (YACs) containing the murine Hox-c cluster. We designed primers specific to the human HOX-C12 and -C13 homeoboxes and used the polymerase chain reaction and sequencing to identify the homeobox of the murine orthologs of these two genes on one of the YACs. Only the YAC containing the 5' end of the Hox-c cluster contained these sequences. Thus, we have identified two mouse homeobox genes not previously reported and established linkage of these genes to the 5' end of the mouse Hox-c cluster.

Animals↗

PubMed1993

Physical linkage of the murine Hox-b cluster and nerve growth factor receptor on yeast artificial chromosomes.

Evidence derived from sequence comparisons and the genomic organization of the murine Antennapedia-class homeobox gene clusters suggest that they arose from a primordial cluster through a process of gene duplication and divergence followed by cluster duplication. A large chromosomal domain surrounding the ancestral homeobox cluster also appears to have been duplicated and has remained relatively stable since the divergence of humans and rodents. To test the extent of the duplicated chromosomal domain, we have initiated physical mapping studies of the regions surrounding the four murine homeobox clusters using pulsed-field gel electrophoresis and yeast artificial chromosome cloning. In this study, we present a long-range restriction map of mouse chromosome 11 spanning 1500 kb in the region surrounding the Hox-b cluster. We have determined that the gene for the nerve growth factor receptor is tightly linked to the Hox-b complex and is located within 50 kb of the Hox-b 1 gene at the 3' end of the cluster. Four yeast artificial chromosomes have been isolated and characterized by the polymerase chain reaction, long-range restriction mapping, and Southern blotting. Two clones of 150 and 300 kb contain the entire Hox-b cluster and the nerve growth factor receptor gene. A 440-kb clone contains the 3' end of the Hox-b cluster, the nerve growth factor receptor gene, and extends downstream. A 210-kb clone contains the 5' end of the Hox-b cluster and extends upstream. These clones confirm the pulsed-field restriction map of uncloned mouse DNA and represent a contig of approximately 600 kb of cloned material from mouse chromosomes 11.

Animals↗

PubMed1991

Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors.

Steroid response elements (SREs) cooperate with many different cis-acting elements including NF-1 sites, CACCC boxes, and other SREs to induce target gene expression (Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90; Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395). Induction of gene expression can be additive or synergistic with respect to the level of activation by either transactivators. Two mechanisms have been proposed for how synergism occurs: 1) cooperative binding of transcriptional activators to DNA or 2) simultaneous interaction of individually bound activators with a common target protein. We have shown previously that cooperative binding of receptors is important for synergism between two progesterone response elements (PREs). Here we showed that an estrogen response element (ERE) and a PRE can also functionally cooperate and this synergism between an ERE and a PRE is not contributed by cooperative DNA binding. Furthermore, we have demonstrated that the activation domains of the progesterone receptor (PR) (C1Act) are required for synergism between two PREs and sufficient for confirming cooperative binding. However these two activation domains of PR are not sufficient for synergism between an ERE and a PRE. Additional regions within the NH2-terminal and COOH-terminal domains are also required for synergistic interaction between two heterologous SREs.

Base Sequence↗

PubMed1990

Generating precise mechanical stimuli and recording chordotonal organ discharge patterns using a microcomputer.

A computer-controlled system for the investigation of the response properties of the tibio-femoral chordotonal organ in the locust is described. The computer is used to generate small amplitude sinusoidal movements of the tibia via a small servo-controlled motor. The resulting response recorded via a suction electrode is simultaneously detected, processed and stored on disk. Full constructional details for all hardware required are given. The software, developed for a BBC microcomputer, in addition to controlling all the hardware, has graphics and analysis routines enabling the operator to display and manipulate the stored data.

Action Potentials↗

PubMed1988

A far upstream ovalbumin enhancer binds nuclear factor-1-like factor.

Band-shifting and DNase I footprinting analyses detected a specific DNA binding protein extracted from oviduct nuclei that binds to the ovalbumin gene 5' sequence between -1094 and -1125. This "-1100" fragment, when inserted upstream of the SV40 or ovalbumin promoters fused to a chloramphenicol acetyltransferase reporter gene, enhances chloramphenicol acetyltransferase activity 5-10-fold following transfection into CV1 cells. The sequence to which the oviduct factor binds contains a nuclear factor-1 (NF-1) half-site (GCCAA). An oligonucleotide matching the sequence of the adenovirus NF-1 binding site competed for binding to the -1100 footprinted region with a higher affinity than an oligonucleotide for the -1100 region itself. Similarly, the -1100 region oligonucleotide also competes for binding of the factor to the NF-1 oligonucleotide. These data suggest that the oviduct factor which binds to the -1100 region is an NF-1-like protein that serves as a steroid hormone-independent enhancer of the ovalbumin gene transcription.

Animals↗

PubMed1988

A steroid response element can function in the absence of a distal promoter.

The glucocorticoid-progesterone responsive element (GRE/PRE) of the tyrosine aminotransferase (TAT) gene is a steroid-inducible enhancer. We show that the GRE/PRE can also work in the absence of a distal promoter element when located 5' to the ovalbumin TATA box. The GRE/PRE in this position retains progesterone or glucocorticoid receptor and hormone dependency for the induction of gene expression. Initiation of transcription occurs correctly, and induction occurs at the mRNA level. These data indicate that a steroid-inducible enhancer can function without a distal promoter element.

Animals↗