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D J Roufa

Publications and source records attributed to D J Roufa.

At least 37 records · Page 2Linked to original sources
PubMed1988

Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

Animals↗
PubMed1987

A cloned human ribosomal protein gene functions in rodent cells.

Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.

Animals↗
PubMed1986

Ribosomal protein gene sequences map to human chromosomes 5, 8, and 17.

DNA sequences complementary to six mammalian ribosomal protein (r-protein) cDNAs are assigned to human chromosomal linkage groups in human-Chinese hamster hybrid cell clones. Ten r-protein DNA fragments map to chromosomes 5, 8 and 17, indicating that these important, housekeeping genes are distributed to multiple sites in the human genome. Each of the chromosome assignments, determined initially by surveying Chinese hamster-human hybrid cell clones with complex karyotypes using Chinese hamster and human cDNA probes, were confirmed in critical minipanels of highly reduced or monochromosomal hybrid cells. As all 10 fragments mapped to only three human chromosomes, r-protein sequences appear to be distributed nonrandomly within human DNA. The r-protein S14 sequence assigned to human chromosome 5 (5q23-5q33) rescues Chinese hamster emetine-resistance mutations (emt b) in interspecific hybrids. Therefore, this sequence corresponds to the transcriptionally active human RPS14 gene. In contrast, other r-protein DNA sequences examined likely are a mixture of functional genes and inactive pseudogenes.

Chromosome Mapping↗
PubMed1986

Homologous ribosomal proteins in bacteria, yeast, and humans.

We describe sequences of two human ribosomal proteins, S14 and S17, and messenger RNAs that encode them. cDNAs were used as molecular hybridization probes to recognize complementary genes in rodent, Drosophila, and yeast chromosomal DNAs. Human ribosomal protein sequences are compared to analogous Chinese hamster, yeast, and bacterial genes. Our observations suggest that some ribosomal protein genes have been conserved stringently in the several phylogenetic lines examined. These genes apparently were established early in evolution and encode products that are fundamental to the translational apparatus. Other ribosomal protein genes examined, although similar enough to heterologous DNA sequences to indicate their structural relationships, appear to have diverged substantially during evolution, probably reflecting adaptations to different genetic environments.

Amino Acid Sequence↗
PubMed1986

Primary structure of human ribosomal protein S14 and the gene that encodes it.

Chinese hamster ribosomal protein S14 cDNA was used to recognize homologous human cDNA and genomic clones. Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical. Two overlapping human genomic S14 DNA clones were isolated from a Charon 28 placental DNA library. A fragment of single-copy DNA derived from an intron region of one clone was mapped to the functional RPS14 locus on human chromosome 5q by using a panel of human X Chinese hamster hybrid cell DNAs. The human S14 gene consists of five exons and four introns spanning 5.9 kilobase pairs of DNA. Polyadenylated S14 transcripts purified from HeLa cell cytoplasma display heterogeneous 5' ends that map within noncoding RPS14 exon 1. This precludes assignment of a unique 5' boundary of RPS14 transcripts with respect to the cloned human genomic DNA. Apparently HeLa cells either initiate transcription at multiple sites within RPS14 exon 1, or capped 5' oligonucleotides are removed from most S14 mRNAs posttranscription. In contrast to the few murine ribosomal protein and several other mammalian housekeeping genes whose structures are known, human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1. Three related short sequence motifs, also observed in murine and yeast ribosomal protein genes, occur in this region of the RPS14 gene. RPS14 introns 3 and 4 both contain Alu sequences. Interestingly, the Alu sequence in intron 3 is located slightly downstream from a chromosome 5 deletion breakpoint in one human X hamster hybrid clone analyzed.

Amino Acid Sequence↗
PubMed1985

Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs.

The Chinese hamster ovary (CHO) cell 40S ribosomal subunit protein S14 provides a unique opportunity to investigate an important mammalian housekeeping gene and its mRNA and protein products. The S14 gene appears to be single copy, and CHO cell S14 mutants have been isolated as emetine-resistant (emtB) clones in tissue culture. Thus, S14 is the only mammalian ribosomal protein whose gene structure and function are amenable to straightforward genetic and biochemical analysis. Recently, we isolated a wild-type Chinese hamster lung cell cDNA clone, pCS14-1, including an almost complete copy of the ribosomal protein S14 message (N. Nakamichi, D. D. Rhoads, and D. J. Roufa, J. Biol. Chem. 258: 13236-13242, 1983). Here we describe comparable cDNAs from wild-type and emtB CHO cells. We report both mRNA and polypeptide sequences of the wild-type and mutant ribosomal protein transcripts. As a consequence of the genetic methods used to obtain our emetine-resistant mutants, the emtB S14 cDNAs differ from wild-type cDNA by single-base changes. Physical and chemical features of polypeptides encoded by the cDNAs are consistent with well-characterized S14 protein polymorphisms. The three emtB mutations analyzed affect two adjacent arginine codons within the very basic S14 carboxyl region, indicating a significant role for this portion of the protein in the function and architecture of the mammalian 40S ribosomal subunit.

Amino Acid Sequence↗
PubMed1983

The Chinese hamster cell emetine resistance gene. Analysis of cDNA and genomic sequences encoding ribosomal protein S14.

We used an intersecting pool strategy to recognize chimeric plasmids containing Chinese hamster ribosomal protein cDNAs. The screening procedure involved hybridization-selection of messenger RNAs, cell-free translation of selected mRNAs, and electrophoresis of polypeptide products on one- and two-dimensional polyacrylamide gels. The protocol was designed to recognize ribosomal protein S14 cDNAs specifically. Of 500 chimeric plasmids screened, two possessed cDNAs complementary to S14 mRNA and 18 contained sequences complementary to other ribosomal protein messages. Previously we demonstrated that mutations affecting Chinese hamster ovary cell ribosomal protein S14 are responsible for genetic resistance to the translational inhibitor emetine (emt b). Because emetine-resistant mutant and wild type Chinese hamster ovary cells elaborate mRNAs that encode electrophoretically distinguishable forms of S14 protein, we were able to identify S14 cDNA clones unambiguously. The data described here indicate that: 1) clone pCS14-1 contains most, if not all, of the S14 coding sequence as a cDNA; 2) S14 mRNA is approximately 0.01% of a Chinese hamster cell's polyadenylated messenger RNA; and 3) genomic DNA-encoding ribosomal protein S14 is a low, perhaps single, copy sequence with a complex structure, including several, long intervening sequences.

Animals↗
PubMed1983

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

Animals↗
PubMed1983

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

Animals↗
PubMed1982

Emetine resistance in chinese hamster ovary cells is associated with an altered ribosomal protein S14 mRNA.

Chinese hamster ovary cell mutants resistant to the translational inhibitor emetine (Emt B mutants) express a single genetically altered 40S ribosomal subunit protein, S14. that appears to account for their drug resistance. To determine whether Emt B mutation affect the structural gene for the S14 protein, we isolated mRNAs from several wild-type and Emt B mutant clones. We translated the mRNAs in a cell-free system derived from wheat germ, and we discerned the biosynthesis of 59 ribosomal proteins, including S14. In every case, poly(A)+ mRNA programmed the cell-free system to synthesize an S14 protein electrophoretically identical to the S14 extracted from the ribosomes of the corresponding cell line. Messages from two Emt B mutants (Emr-2 and Emr-2-2) specified S14s that were electrophoretically distinct from the wild-type protein. Thus, Emt B mutations were expressed in mutant cell mRNAs, apparently reflecting mutagen-induced changes in S14 structural genes.

Animals↗
PubMed1982

Nucleosome segregation at a defined mammalian chromosomal site.

When animal cells replicate chromatin under conditions precluding new histone biosynthesis, half of the daughter DNAs are devoid of nucleosomes and are sensitive to staphylococcal nuclease. DNA sequences resistant to nuclease are associated with preexisting nucleosomes, which redistribute to progeny DNA duplexes during replication. We labeled newly replicated DNA sequences in a simian virus 40 (SV40)-transformed Chinese hamster cell clone with 5-bromodeoxyuridine (BrdUrd) in the presence and absence of a protein biosynthesis inhibitor, emetine. We resolved single-stranded BrdUrd- and dT-DNA sequences protected from nuclease digestion by nucleosomes and determined from which strands of the integrated viral DNA parental template (dT) and newly replicated progeny (BrdUrd) sequences were derived. Because we knew that the cell clone studied contained all of its integrated SV40 DNA at a single chromosomal site, we were able to determine that preexisting nucleosomes segregated to only one of the two daughter duplexes containing the integrated viral sequence. Additionally, in the presence of emetine, the integrated viral origin of replication, ORIsv, appeared not to function as a chromosomal replication origin, perhaps reflecting the drug's effect on synthesis of SV40 large tumor antigen.

Cell Transformation, Viral↗
PubMed1981

Replication of viral DNA sequences integrated within the chromatin of SV40-transformed Chinese hamster lung cells.

To determine when during S phase integrated viral DNA sequences in several tsA SV40-transformed Chinese hamster cell clones replicate, we pulse-labeled cultures with BrdUrd and subsequently collected mitotic cells during sequential time intervals. Restriction endonuclease mapping indicated that each of the three SV40-transformed Chinese hamster lung cell clones contained a single viral DNA sequence at a different, but in each case unique, chromosomal site. DNA was extracted from each population of mitotic cells and then was resolved into dense, BrdUrd-containing and light, unsubstituted DNA fractions by cesium chloride gradient centrifugation. In each pair of samples obtained, we measured viral DNA sequences by solution hybridization using single-stranded SV40 32P-labeled DNA probes. Our results support the conclusions that specific genes within a mammalian DNA are programmed to replicate at particular times during S phase, and that the SV40 A gene product, large T antigen, programs integrated viral DNA sequences to replicate very early in S phase. The fractions of viral DNA replicated early in S phase appeared to correlate with each clone's content of functional large T antigen at permissive and nonpermissive culture temperatures.

Animals↗
PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗
PubMed1979

A ts mutant isolated from CHL cells: inhibition of DNA replication at nonpermissive temperature.

A mutant Chinese hamster cell clone has been isolated from an S phase culture. This clone, ts154, could not replicate DNA at nonpermissive temperature (39 degrees C), although it synthesized DNA normally at permissive temperature (33 degrees C). After transfer to 39 degrees, ts154 cells replicated DNA semiconservatively through precisely two complete rounds and divided twice before they ceased DNA synthesis and further growth. The mutation in ts154 affects DNA replication specifically, as its rates of cellular transcription, its ability to support growth of vesicular stomatitis virus, and its ribo- and deoxyribonucleoside triphosphate pools were normal at 39 degrees. Cultures of ts154 resumed DNA replication within 6h when shifted from 39 degrees to 33 degrees, even after 78 h at the nonpermissive temperature. Upon return to 33 degrees, mutant cells achieved a normal steady-state rate of DNA synthesis by 10 h, suggesting that they had come to rest within G1 phase at 39 degrees. Resumption of DNA synthesis and growth after a shift-down to 33 degrees were sensitive to cycloheximide in ts154, and thus likely required synthesis of new protein.

Animals↗
PubMed1979

Emetine resistance in Chinese hamster cells is linked genetically with an altered 40S ribosomal subunit protein, S20.

Emr-2 is an emetine-resistant (Emr) Chinese hamster ovary cell mutant that contains a single electrophoretically altered ribosomal protein, a component of its 40S ribosomal subunit [Boersma, D., McGill, S., Mollenkamp, J. & Roufa, D.J. (1979) J. Biol. Chem. 254, in press]. This report describes a genetic experiment designed to test linkage between the genes that specify the altered ribosomal protein, S20*, and emetine resistance in Emr clones that segregated from cultures of Emr-2 cells hybridized with emetine-sensitive cells. The data described indicate that Emr and S20* phenotypes are due to mutations linked to the same chromosome in the Chinese hamster genome; most likely they are due to the same mutation. The data also confirm earlier speculations by others that the Emr locus in Chinese hamster cells is hemizygous.

Animals↗
PubMed1976

5-bromodeoxyuridine-DNA strand symmetry and the repair of photolytic breaks in Chinese hamster cell chromosomes.

Experiments described in this report quantiate the black light sensitivities of Chinese hamster lung (CHL) cells containing BrdUrd-DNA of defined composition. Cesium chloride equilibrium gradient centrifugation provides estimates both of the percent thymidine replacement by BrdUrd and of the symmetry (unifilar versus bifilar) of BrdUrd incorporation into the chromosomal DNA duplexes. Radiation damage to BrdUrd-substituted CHL cell DNA and its repair in situ also have been assessed by alkaline sucrose gradient sedimentation. We observe that animal cell sensitivities to visible light (300-400 nm wavelength) depend markedly upon the symmetries of BrdUrd-substitution within the cells Dna. Cells that contain only unifilar BrdUrd-DNA are resistant to black light, whereas cells that contain bifilar BrdUrd-DNA are extremely photosensitive. The former cell populations repair single-stranded nicks (breaks in phosphodiester bonds) in their DNA within 24 hr of irradiation; the latter cell populations, however, are not able to repair light-induced, double-stranded breaks in their DNA.

Bromodeoxyuridine↗