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Biomedical subjects

M C Rice

Publications and source records attributed to M C Rice.

At least 19 recordsLinked to original sources
PubMed2001

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides.

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.

Base Sequence↗
PubMed2001

The potential of nucleic acid repair in functional genomics.

Chimeric RNA/DNA oligonucleotides have been used successfully to correct point and frameshift mutations in cells as well as in animal and plant models. This approach is one of several nucleic acid repair technologies that will help elucidate the function of newly discovered genes. Understanding the mechanisms by which these different technologies direct gene alteration is essential for progress in their application to functional genomics.

DNA↗
PubMed2001

In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae.

Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has proven successful in eukaryotic cells including animal and plant models. In many cases, however, there has been a disparity in the levels of gene correction or frequency. While the delivery of these chimera into the nucleus and the long-term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the key to improving or stabilizing frequency. To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experiments in cell-free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors. RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppressive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, containing a marker gene, were converted to wild type, evidenced by the expression of green fluorescence in converted cells. Because the repaired fusion gene contains a corrected neomycin sequence, cells were subsequently placed under G418 selection and conversion confirmed at the genetic level. Taken together, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for evaluating true frequencies of correction. Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling measurements of conversion in real time.

Base Sequence↗
PubMed2000

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts.

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Animals↗
PubMed2000

Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides.

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.

Base Sequence↗
PubMed1999

Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can Be partially rescued in a p53(-/-) background.

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.

Animals↗
PubMed1998

Analysis of the human RAD51L1 promoter region and its activation by UV light.

The human REC2/RAD51B gene (HGMW-approved symbol RAD51L1) encodes a 350-amino-acid protein with regional homologies to members of the RAD52 epistasis group. It is induced by DNA-damaging agents, and the overexpression of this gene product causes G1/S cell cycle arrest. In this report, the promoter region, containing the UV-responsive element, is revealed. Deletion analyses of a 1699-base fragment at the 5' end of the human REC2/RAD51B cDNA identified a 116-base sequence that appears to be responsible for radiation induction. This fragment contains many DNA sequences that have been identified in the promoter regions of other radiation-inducible genes in yeast and humans. Within this region are "consensus" binding sites for both the AP2 and the p53 proteins that may act to regulate the expression of the human REC2/RAD51B gene. Five putative transcripts have been identified from regions 5' of the promoter element that splice near the ATG translation start site. None of the transcripts contain the UV-inducible element nor the consensus transcription factor binding sites.

Base Sequence↗
PubMed1998

The human REC2/RAD51B gene acts as a DNA damage sensor by inducing G1 delay and hypersensitivity to ultraviolet irradiation.

HsRec2/Rad51B is a 350-amino acid protein with a molecular mass of 38,300 Da that appears to be involved in cell cycle regulation and UV-induced apoptosis. The mouse and human genes were isolated based on their homology to a recombinational repair gene from Ustilago maydis and contain functional domains to hRAD51 and hLIM 15 (M. C. Rice et al., Proc. Natl. Acad. Sci. USA, 94: 7417-7422, 1997). Here, we report the results of studies on the behavior of CHO cells containing a plasmid encoding a wild-type hsRec2/Rad51B, a full-length protein with a single mutation at residue 163, which lies in the putative src site, and a truncated version of hsRec2/Rad51B, containing only the first 100 amino acids at the NH2 terminus. Using fluorescence-activated cell sorting analysis to follow the progression of cells through the cell cycle, we find that stable transfectants constitutively overexpressing the wild-type human Rec2/Rad51B protein exhibit a G1 delay. In addition, when irradiated with UV at a dose of 15 J/m2, CHO cells transfected with the various hREC2/RAD51B vectors exhibited different responses. Cells expressing the wild-type human Rec2/Rad51B underwent apoptosis, with the greatest cell death occurring 24 h after irradiation. The control cells, which contained an empty vector, and the cells expressing truncated hsRec2/Rad51B or the full-length Rec2 with a mutation at residue 163 did not. In summary, these findings of cell cycle slowing and UV-induced apoptosis in CHO cells constitutively expressing the human Rec2/Rad51B protein suggest that hsRec2/Rad51B plays a role in a DNA damage surveillance pathway.

Animals↗
PubMed1997

Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis.

A human and a mouse gene have been isolated based on homology to a recombinational repair gene from the corn smut Ustilago maydis. The new human (h) gene, termed hREC2, bears striking resemblance to several others, including hRAD51 and hLIM15. hREC2 is located on human chromosome 14 at q23-24. The overall amino acid sequence reveals characteristic elements of a RECA-like gene yet harbors an src-like phosphorylation site curiously absent from hRAD51 and hLIM15. Unlike these two relatives, hREC2 is expressed in a wide range of tissues including lung, liver, placenta, pancreas, leukocytes, colon, small intestine, brain, and heart, as well as thymus, prostate, spleen, and uterus. Of greatest interest is that hREC2 is undetectable by reverse transcription-coupled PCR in tissue culture unless the cells are treated by ionizing radiation.

Amino Acid Sequence↗
PubMed1997

Interaction between Ustilago maydis REC2 and RAD51 genes in DNA repair and mitotic recombination.

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated, rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and gamma radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.

Amino Acid Sequence↗
PubMed1996

Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide.

A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin betaS allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the betaS mutation, there was a detectable level of gene conversion of the mutant allele to the normal sequence. The efficient and specific conversion directed by chimeric molecules may hold promise as a therapeutic method for the treatment of genetic diseases.

Alleles↗
PubMed1980

Genetic variance of laboratory outbred Swiss mice.

The extent of allelic variation has been estimated at 46 structural gene loci within three major colonies of Swiss mice and between inbred derivative strains. The colonies have retained nearly the same amount and type of variation found in natural murine or human populations despite laboratory propagation for more than 50 years (175 generations). The population genetic structures of the Swiss mouse colonies were comparable to an island population in which random fixation, and not inbreeding or population bottlenecks, is apparently responsible for slight losses in genetic variance.

Alleles↗
PubMed1980

Genetic diversity in leukemia-prone feral house mice infected with murine leukemia virus.

The Lake Casitas (LC) mouse population located in south western Ventura county in California is unusual insofar as 85% of these mice are persistently viremic with congenitally transmitted murine leukemia virus (MuLV). The virus has been identified as the etiological agent responsible for lymphoma and neuromotor paralysis in large numbers of the mice. The majority of other wild mouse populations are generally free of infectious MuLV despite the presence of endogenous cellular DNA sequences homologous to infectious virus isolated from wild mice. Electrophoretic variation in 46 gene-enzyme systems was surveyed using mice from Lake Casitas and from a virus-negative population located in Bouquet Canyon (BC) approximately 40 miles from Lake Casitas. The LC and BC populations are genetically very similar to each other and to feral mouse populations previously studied in California and Europe. In the LC population 24% of the loci are polymorphic compared to 17% in the BC population. The average heterozygosities for the LC and Bc populations are 0.094 and 0.073, respectively. The large amount of genic variation in LC fails to support the concept of the derivation of the colony from a small number of founders. Tests for linkage disequilibrium and/or selective association of viremia and polymorphism at 15 loci located on nine mouse chromosomes did not reveal any nonrandom assortments. The viremic LC population, then, appears indistinguishable within the limits of experimental resolution from the virus-negative BC population in its population genetic structure.

Animal Population Groups↗
PubMed1977

Studies of urticaria and acute serum sickness with the C1q precipitin test.

The C1q precipitin test was performed in serum samples from five groups of patients: (1) 20 patients with acomplementemic systemic lupus erythematosus glomerulonephritis (SLE), (2) 2 patients with serum sickness due to the administration of horse serum, (3) 2 patients with serum sickness preceding hepatitis B, (4) 50 patients with chronic urticaria, and (5) 30 normal controls. Positive C1q precipitin tests were found in all patients with SLE and the four cases of serum sickness. Positive tests correlated with depressed serum complement (C3 and C4) levels and were found only in the early phase of serum sickness. Urticaria patients uniformly had negative C1q precipitin tests and normal serum complement levels.

Acute Disease↗
PubMedDate not supplied

In vitro complement consumption by contrast materials and analogues: reactors vs. nonreactors.

Blood samples from previous contrast reactors and nonreactor controls were incubated with diatrizoate and several contrast analogues. Total complement levels were assayed. All the agents caused complement activation generally proportional to their concentration. Reactors' sera responded to lower concentrations of contrast or analogues than did control sera. Such studies might be of value in predicting contrast reactors.

Acetamides↗