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G E Roth

Publications and source records attributed to G E Roth.

8 recordsLinked to original sources
PubMed1991

Replication analysis of plasmid DNAs injected into Drosophila embryos.

From a "shotgun" collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and non-methylation. Within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.

Animals↗
PubMed1987

["Artificial" chromosomes].

The structural elements required for chromosome replication, segregation, and stability are replication origins, centromeres, and telomeres. DNA sequences capable of organizing these three elements have been isolated from yeast chromosomal DNA by means of recombinant DNA techniques and yeast cell transformation. It is now possible to combine these sequences into "artificial" chromosomes for yeast cells to obtain more insight into chromosome structure and function. Evidence is presented that the construction of artificial chromosomes functional in higher eukaryotes will be possible in the near future.

Base Sequence↗
PubMed1985

Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.

Age Factors↗
PubMed1983

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.

Animals↗
PubMed1981

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum.

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33 258 -- CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 x 10(5) copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a 'theme and variation' pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.

Animals↗
PubMed1979

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodes Ascaris suum and Parascaris equorum.

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres ("chromatin diminution", Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species, Parascaris equorum var. univalens and Ascaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA of P. equorum and A. suum has the buoyant density of 1.697 g/cm3, while soma DNA of both species bands at 1.700 g/cm3. In P. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm3, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs of P. equorum and A. suum the analysis of their base composition revealed differences (40% guanine plus cytosine in P. equorum and 36% in A. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of total A. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement of Ascaris does not follow the short period interspersion pattern observed in most organism.

Animals↗