Search PubMed⌕ Search

Biomedical subjects

R H Reeder

Publications and source records attributed to R H Reeder.

At least 73 records · Page 4Linked to original sources
PubMed1984

The mechanism of nucleolar dominance in Xenopus hybrids.

We have mimicked the phenomena of nucleolar dominance by injecting cloned ribosomal genes from X. laevis and X. borealis into oocytes from either species and into embryos. The results of these experiments suggest that the dominance of the laevis ribosomal genes in hybrid embryos is primarily due to the fact that the laevis spacer has more copies of an enhancer element for the ribosomal gene promoter than does the borealis spacer. A contributing effect is that the laevis promoter functions well with transcription machinery from either species while the borealis promoter interacts poorly with laevis transcription machinery.

Animals↗
PubMed1984

On the mechanism of nucleolar dominance in mouse-human somatic cell hybrids.

The mechanism of nucleolar dominance was studied in two lines of mouse-human somatic hybrids. Both lines had preferentially lost human chromosomes but had retained significant amounts of both mouse and human ribosomal genes (genes coding for the 18S, 5.8S, and 28S RNAs of ribosomes). However, the human ribosomal genes were repressed, and only mouse ribosomal genes were expressed. Soluble transcription extracts from the hybrids were able to initiate RNA synthesis accurately on a cloned mouse ribosomal gene but were unable to initiate accurately on a human ribosomal gene. This suggests that nucleolar dominance in these hybrids is due to the loss or inactivation of the gene for a specificity factor required to recognize the human ribosomal gene promoter.

Animals↗
PubMed1984

Effect of intercalating agents on RNA polymerase I promoter selection in Xenopus laevis.

We have analyzed the effect of DNA intercalating agents on the transcription signals from two different Xenopus laevis RNA polymerase I promoters. The transcription signal from the promoter for the 7.5-kilobase rRNA precursor (the gene promoter) is unaffected over a large range of intercalating agent concentrations regardless of whether the template is injected plasmid DNA in oocytes, the amplified endogenous nucleoli of oocytes, or the endogenous chromosomes of cultured Xenopus kidney cells. The transcription signal from a closely related promoter located in the spacer DNA between genes (the spacer promoter) ranges between undetectable to equivalent to the gene promoter signal on different templates. The transcription signal from the spacer promoter is also differentially affected by intercalating agents relative to the gene promoter. Depending on the template, these agents can either increase or decrease the transcription signal from the spacer promoter. Fusions between the gene and spacer promoters demonstrate that intercalating agents affect transcription initiation. One explanation for these results is that the degree of supercoiling of the template DNA can differentially inhibit transcription from the spacer promoters. The different effects of intercalating agents on transcription from the spacer promoters of various templates could then be explained as differences in the degree of supercoiling present on these templates initially.

Animals↗
PubMed1984

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

Animals↗
PubMed1983

Spacer regulation of Xenopus ribosomal gene transcription: competition in oocytes.

Xenopus laevis ribosomal gene plasmids bearing different length spacers were injected into oocyte nuclei in competition with each other. The spacer has two basic effects on transcription from the gene promoter. First, if the competing pair have unequal spacer lengths, the gene promoter attached to the longer spacer is always dominant in transcription (the competition effect). Second, as the total amount of spacer in the reaction increases, the total amount of transcription decreases (the sink effect). Both the competition and the sink effect are attributed to sequence elements that are 60 or 81 bp long, which are present in multiple copies in the spacer and are related in sequence to part of the gene promoter. The 60/81 bp elements confer competitive dominance in either orientation. A model which explains both the competition and the sink effects is discussed in which the 60/81 bp elements are attraction sites for a factor(s) which is needed to activate the gene promoter.

Animals↗
PubMed1983

Nested control regions promote Xenopus ribosomal RNA synthesis by RNA polymerase I.

The DNA regions required for accurate initiation by RNA polymerase 1 on the ribosomal RNA genes of Xenopus laevis consist of at least two domains within a 150 bp segment adjoining the initiation site. A region of only 13 nucleotides (from -7 to +6) appears sufficient to specify accurate and efficient initiation when various 5' and 3' deletion mutants are injected into oocyte nuclei. In contrast, a 10-fold larger region (from -142 to +6) is required for maximal synthesis when deletion mutants are assayed in oocyte nuclear homogenates; only a low level of synthesis is specified by the 13 bp promoter domain in this in vitro system. Site-specific mutants demonstrate that a sequence adjoining nucleotide -75 is also essential for efficient in vitro initiation. Under other conditions, transcription can be strongly affected by sequences that extend several kb upstream in the rDNA spacer and contain duplications of sequences found within the 150 bp promoter. We propose a model in which transcription of the X. laevis rRNA genes is regulated by interaction of at least three sequence domains that extend over a several kb region.

Animals↗
PubMed1983

Spacer sequences regulate transcription of ribosomal gene plasmids injected into Xenopus embryos.

We have injected plasmids containing a repeating unit (spacer plus gene) of Xenopus laevis ribosomal DNA into fertilized eggs. Transcription on these plasmids begins at the same time as transcription on the endogenous ribosomal genes (late blastula stage). Previous work defined the ribosomal gene promoter as the region from -140 to +6 around the site of transcription initiation (Reeder et al., 1982; Moss, 1982; Sollner-Webb et al., submitted). We show here that the level of transcription of the injected ribosomal genes is strongly affected by spacer sequences far upstream of the promoter. Deletion of spacer sequences over 1150 bp from the initiation site reduces the transcription signal from injected plasmids by a factor of 5-10. We propose that the upstream spacer sequences act to influence the frequency of promoter activation.

Animals↗
PubMed1983

Transcription in cloned spacers of Xenopus laevis ribosomal DNA.

Rare individuals of Xenopus laevis exhibit frequent initiation of transcription in the spacers of oocyte ribosomal DNA (rDNA). Using electron microscopy we have characterized spacer transcription in such an individual and have confirmed that the sites of transcription initiation correspond to the imperfectly duplicated promoters ("Bam islands") present in the X. laevis rDNA spacer. We have cloned a repeat unit containing a gene and a spacer from this individual and have injected the recombinant plasmid, pXlr 164, into oocytes of other X. laevis individuals. In electron microscope preparations the spacers of some of the cloned repeats were transcribed by RNA polymerase I. This demonstrates that the ability to initiate transcription at the Bam islands is a property of the spacer DNA. On pXlr 164, initiation in the spacer occurred about 5% as frequently as transcription from the gene promoter. However, transcribed spacers were as closely packed with RNA polymerase as was the gene. We conclude that polymerase I promoters may vary over a wide range in the frequency with which they "activate" but that once activated all can load polymerases to maximal density. The presence or absence of spacer transcription had no observable effect on either the frequency of activation or the density of polymerase loading of the gene immediately downstream. A subclone, pXlr 264, containing only spacer DNA also showed regular initiation and termination, providing further evidence that there is an effective "fail-safe" termination signal 225 base pairs upstream from the rRNA gene initiation site.

Animals↗
PubMed1982

Mapping of transcription initiation and termination signals on Xenopus laevis ribosomal DNA.

We have injected cloned derivatives of Xenopus laevis ribosomal genes into X. laevis oocyte nuclei and examined the resulting transcription complexes in the electron microscope. From this work we conclude that the promoter lies somewhere within a region between -320 nucleotides upstream and +113 nucleotides downstream from the site of transcription initiation. This assignment agrees with inferences based on sequence conservation. It further suggests that the duplicated initiation region sequences located further out in the spacer ("Bam islands") are not required for the normal high densities of RNA polymerase loading seen on ribosomal genes. Concerning termination, the cluster of four Ts that forms part of the HindIII restriction site at the 3' end of the gene appears to be part of the normal termination signal. Termination still occurs when only three Ts are present, but reduction to two Ts damages termination. Because clusters of three Ts appear at several sites within the gene, it is likely that sequences adjacent to the T cluster also are required for normal termination. In addition, we present evidence for a fail-safe termination site just upstream from the site of transcription initiation.

Animals↗
PubMed1980

Initiation of ribosomal RNA chains in homogenates of oocyte nuclei.

We have studied RNA synthesis in homogenates of nuclei manually isolated from mature oocytes of Xenopus laevis. Transcription of the endogenous template is completely resistant to alpha-amanitin and represents primarily transcription of the amplified ribosomal genes. Three lines of evidence demonstrate that RNA polymerase I is reinitiating RNA chains in this system. First, transcription continues in the homogenates for at least 8 h. During this time, 50 to 100 RNA copies are synthesized per gene. Second, inhibitors of initiation, such as heparin and sodium dodecyl sarcosinate, cause synthesis to plateau within 60 min with less than 15 RNA copies synthesized per gene. Third, incorporation of gamma-thio nucleoside triphosphates demonstrates that reinitiation is occurring. When RNA is synthesized in the presence of adenosine-5'-O-(gamma-thiotriphosphate) and guanosine-5'-O-(gamma-thiotriphosphate), 3 to 10% of the newly made RNA binds to a Hg-agarose affinity column. After 7.5 min of synthesis the gamma-thio-initiated RNA binding to Hg-agarose hybridizes exclusively to a restriction fragment contining the first 2250 base pairs of the gene. At longer times of synthesis, the gamma-thio-initiated RNA hybridizes more and more to a restriction fragment located further down the gene. The length distribution of this RNA on gels is consistent with its hybridizaton behavior and these data suggest an in vitro chain growth rate of 2 to 5 nucleotides/s. All of the gamma-thio-initiated RNA hybridizes to the H or coding strand of rDNA. The results are consistent with initiation in vitro occurring at the same site used in vivo. Addition of cloned ribosomal genes to the system causes no stimulation of transcription until it is added in a 4000-fold excess over the endogenous ribosomal genes. At that excess it is symmetrically transcribed by RNA polymerase III.

Adenosine Triphosphate↗
PubMed1979

The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis.

In this study, we have located the sites of transcription initiation and termination on a cloned fragment of ribosomal DNA from X. laevis, and have sequenced the surrounding nucleotides. As reported previously (Reeder, Sollner-Webb and Wahn, 1977), about 25% of the 40S rRNA precursor molecules isolated from oocytes have polyphosphate 5' termini and are therefore presumed to represent primary transcripts. These ends hybridize specifically to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a 221 bp fragment of ribosomal DNA. The nucleotides encoding the 5' end of the 40S RNA were located more precisely by two methods. In one, we hybridized 40S RNA to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a smaller DNA fragment and extended the recessed 3' terminus of the DNA using reverse transcriptase. The resultant DNA fragments were sized on sequencing gels. Both determinations map the 5' end of 40S RNA at the same site in the rDNA, about 2250 bp upstream from the Eco RI site in the 18S rRNA coding sequence. At this site we find a DNA sequence beginning AGGGGAAGAC.... which agrees with partial sequence data from the 5' end of polyphosphorylated and bulk 40S rRNA. Features of this region of the ribosomal DNA will be discussed in this paper. A 227 nucleotide region surrounding the initiation site was also sequenced from an independently derived clone and found to differ in only one nucleotide. In addition, a sequence is found about 1100 nucleotides upstream from the 5' end of the gene that has 90% homology to the sequence from nucleotides minus 125 to +4 in the initiation region. At the termination region, X. laevis ribosomal DNA has a single recognition site for the restriction enzyme Hind III in each repeating unit. Using the S1 nuclease technique, the 3' termini of both the 40S precursor and mature 28S rRNA are seen to map within this recognition sequence. The sequence surrounding the Hind III site has striking homology to termination sites recognized by other RNA polymerase classes. Sequences with similar features are also found upstream from the initiation site.

Animals↗
PubMed1979

Partial mapping of methylated sequences in Xenopus laevis ribosomal RNA by preparative hybridization to cloned fragments of ribosomal DNA.

Xenopus laevis rRNA was hybridised to either of two cloned fragments of ribosomal DNA. One fragment, designated X1r11, contains a short region of the 18 S rRNA gene and most of the 28 S rRNA gene. The other fragment, X1r14, contains a short region of the 28 S gene and most of the 18 S gene. After hybridization the non-complementary rRNA was removed by digestion with T1 RNase and the hybridized RNA was then eluted and examined by fingerprinting analysis. The 3' terminal sequence and the dimethyl-A-containing sequence of 18 S rRNA both hybridized to X1r11 rDNA, in agreement with the known direction of transcription of rDNA. The distribution of other methylated oligonucleotides between the various fingerprints permitted assigment of nearly all of the methylated sequences in 18 S and 28 s rRNA to either the short 3' region or the long 5' region of the respective molecules.

Animals↗
PubMed1978

DNA synthesis in a multi-enzyme system from Xenopus laevis eggs.

Cytoplasm from unfertilized eggs of the frog Xenopus laevis was separated by DEAE-cellulose column chromatography into nine fractions. Supercoiled pXir 11 DNA molecules (pXir 11 is a Col El-based recombinant plasmid containing part of the Xenopus laevis 18S and 28S ribosomal genes and transcribed spacer region) were incubated with each fraction singly and in various combinations. After incubation for 4 hr at 26 degrees C, the pXir 11 DNA was reisolated and examined by electron microscopy. Using appropriate reaction conditions (pH 7.2, 10 mM Mg2+, 250 micron NTP, 50 50 micron dNTP, 50 MM KCl, fractions III and IV or VI), at least 5-10% of the input DNA was converted to theta structures (presumed intermediates in DNA replication).

Animals↗