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M B Rogers

Publications and source records attributed to M B Rogers.

31 records · Page 2Linked to original sources
PubMed1993

Cloning and characterization of the endogenous cephalosporinase gene, cepA, from Bacteroides fragilis reveals a new subgroup of Ambler class A beta-lactamases.

Bacteroides fragilis CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. beta-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this beta-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The beta-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A beta-lactamase-deficient mutant strain of B. fragilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal beta-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other beta-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of beta-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A beta-lactamases.

Amino Acid Sequence↗
PubMed1993

An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells.

The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage.

Animals↗
PubMed1992

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.

Bacteroides fragilis↗
PubMed1992

Bone morphogenetic proteins-2 and -4 are involved in the retinoic acid-induced differentiation of embryonal carcinoma cells.

Bone morphogenetic proteins-2 and -4 (BMPs-2 and -4) are transforming growth factor beta-related proteins that can induce bone formation in vivo. We observed that the level of endogenous BMP-2 mRNA increased an average of 11-fold on differentiation of F9 embryonal carcinoma cells into parietal endoderm after treatment with retinoic acid (RA) and cAMP, whereas the message for the closely related BMP-4 decreased 12-fold after this treatment. Therefore, the effects of exogenous recombinant BMP-2 protein on the RA-induced differentiation of F9 embryonal carcinoma cells were investigated. BMP-2 addition altered the growth and morphology of RA-treated but not untreated cells. Moreover, the abundance of several messages was affected by exogenous BMP-2 treatment. Notably, the BMP-2 and -4 messages themselves were reduced by the addition of exogenous BMP-2. The observations suggest that RA, which is known to affect bone morphogenesis, may regulate the osteoinductive proteins, BMP-2 and -4. Furthermore, BMP-2 and -4 may be involved in preimplantation embryogenesis.

Animals↗
PubMed1991

The oncofetal gene Pem encodes a homeodomain and is regulated in primordial and pre-muscle stem cells.

The oncofetal gene, Pem, is expressed in a stage specific manner during murine ontogeny. The carboxy terminal portion of the predicted Pem protein has significant similarity to homeodomains of the Drosophila prd family. The Pem gene is expressed in undifferentiated embryonal stem (ES) and embryonal carcinoma (EC) cell lines. Pem mRNA is induced 35-fold in ES cells differentiated in the absence of retinoic acid. Pem mRNA is increased in EC cells differentiated towards parietal or visceral endoderm, consistent with the abundant Pem expression in embryonic yolk sac. In 10T mesenchymal stem cells committed to muscle cell differentiation, Pem mRNA expression is dramatically increased. The elevation in Pem expression preceded the induction of the muscle master regulatory gene, myoD. We conclude that the Pem gene encodes a candidate transcription factor which is developmentally regulated.

Amino Acid Sequence↗
PubMed1991

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes.

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

Animals↗
PubMed1990

Plasmid transformation of Bacteroides spp. by electroporation.

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.

Bacteroides↗
PubMed1990

Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation.

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.

Animals↗
PubMed1990

Region-specific expression in early chick and mouse embryos of Ghox-lab and Hox 1.6, vertebrate homeobox-containing genes related to Drosophila labial.

A chick gene homologous to the Drosophila homeobox gene labial has been cloned and sequenced. Regions of additional sequence identity outside of the homeobox reveal a close relationship to the mouse gene Hox 1.6. Northern blot analysis demonstrates that Ghox-lab and Hox 1.6 transcripts are both present at high levels during early stages of chick and mouse development, with a subsequent decline in abundance to very low levels by the time limb mesenchyme begins to differentiate. In situ hybridization analysis of chick embryos shows intense expression of Ghox-lab mRNA by Hamburger and Hamilton stage 4 (avian 'mid gastrula') and by stage 6 (pre-somitic neural plate) with expression decreasing shortly thereafter. The pattern of Ghox-lab RNA expression in these early embryos divides the embryo into an anterior and a posterior compartment. At stage 6, considerable signal is observed in the posterior two thirds of the embryo, while none is detected in the anterior third which is fated to become the head. This pattern is purely regional in nature, and does not follow boundaries defined by known tissue types. In situ hybridization of Hox 1.6 probes to mouse embryos of day 7.5 or 8.0 indicate that the Hox 1.6 transcript has a temporal and spatial distribution very similar to that of Ghox-lab in the chick embryo.

Amino Acid Sequence↗
PubMed1989

Cloning of Tetrahymena genomic sequences whose message abundance is increased during conjugation.

A molecular and biochemical inquiry into protein regulation during Tetrahymena thermophila conjugation was carried out in two ways: a two-dimensional gel analysis of newly translated proteins and the molecular cloning of genes whose message abundance is increased. The two-dimensional gel analysis indicated that the synthesis of 32 predominantly basic proteins was stimulated in conjugating cells. The induction of these proteins could not be correlated with length of starvation or with mating type. The transcription pattern and molecular organization of three clones of T. thermophila genomic DNA, selected on the basis of differential hybridization to conjugating or control cell RNA, were investigated. Two of the clones, which were homologous to transcripts detected in conjugating cells, showed no rearrangements between micro- and macronuclear DNA. A third clone was divided into three segments. One segment was homologous to sequences limited to the micronucleus. A second segment hybridized to a large number of restriction fragments of micronuclear DNA digested with HindIII but to only two fragments of macronuclear DNA. A third segment, which was complementary to one transcript in conjugating cells and to two different transcripts in control cells, hybridized to two fragments in micronuclear DNA and one fragment in macronuclear DNA.

Animals↗
PubMed1985

Adolescence in Tetrahymena thermophila.

The development of sexual maturity in Tetrahymena thermophila has been shown to include an intermediate stage, adolescence, during which cells are capable of mating with mature cells but not other adolescent cells. When the progeny of successfully mated cells are grown logarithmically and tested frequently for the ability to mate, they are unable to form mating pairs for about 65 generations. This period is known as immaturity. During the next stage, the progeny pair with mature cells but not with other adolescent cells despite the presence of complementary mating types. Adolescence persists for 20-25 generations before the cells attain maturity, which is defined as the ability to mate with any cell of different mating type. Once paired with mature cells, adolescents successfully complete conjugation. Cytological preparations show that both members of the pair undergo meiosis and form macronuclear anlagen. The proteins synthesized during a mating between adolescents and mature cells are similar to those synthesized during a mating between mature cells as determined by two-dimensional gel analysis. Both the adolescent cell and the mature partner contribute genetic markers to the progeny.

Animals↗