Less repair of pyrimidine dimers and single-strand breaks in genes by scid cells.

Severe combined immunodeficient (Scid) mice have a mutation in the catalytic subunit of the DNA binding protein kinase that is involved in repair of double-strand breaks in DNA. To determine if the protein also influences repair of single-strand breaks, we examined the ability of Scid cells to repair lesions introduced by ultraviolet light and gamma-ray irradiation. DNA repair was measured both in total genomic DNA and in specific genes from murine Scid and wildtype fibroblast cell lines. The removal of pyrimidine dimers and repair of strand breaks in genes was measured using quantitative Southern blot analyses. After ultraviolet irradiation, there was no significant difference in the repair of photoproducts in bulk DNA between Scid and wildtype cells, as measured by cellular survival and unscheduled DNA synthesis. However, deficient repair was evident in genes, where Scid cells had 25-50% less repair in the c-myc and dihydrofolate reductase genes. After gamma-irradiation, Scid fibroblasts had 20-35% less repair of DNA breaks in immunoglobulin kappa and heavy constant genes than wildtype cells. The data suggest that intact DNA-PK enzyme is needed for the efficient operation of cellular repair of pyrimidine dimers and single-strand breaks in genes, as well as in its established role in rejoining double-strand breaks.

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein.

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.

Quantitative trait locus analysis of fatty acid concentrations in maize.

A study was conducted to determine the number and chromosomal location of quantitative trait loci (QTL) influencing the concentration of five fatty acids in 200 F2S1 lines derived from an Illinois High Oil (IHO) by Illinois Low Oil (Early Maturity) (ILO(EM)) cross. Restriction fragment length polymorphism (RFLP) analysis was performed on the 200 S1 lines and concentrations of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were determined in self-pollinated kernels harvested from plants grown in replicated field trials during 1992 and 1993. A series of 74 cDNA and genomic clones were used and these revealed 80 polymorphic loci spaced, on average, 24 cM apart throughout the maize genome. Analysis of variance detected significant (p < 0.05) associations between several RFLP loci and the concentration of each fatty acid. A total of 15 RFLP loci clustered in 12 chromosomal regions were associated with the concentration of 16:0, 17 loci clustered in 10 regions were associated with the concentration of 18:0, 12 loci clustered in eight regions were associated with the concentration of 18:1 and 18:2, and 17 loci clustered in eight regions were associated with the concentration of 18:3. Multiple linear regression models consisting of four RFLP loci explained 24 and 62% of the total phenotypic and genotypic variation (R2) among the 200 F2S1 lines for 16:0, five loci explained 51 and 71% of the variation for 18:0, three loci explained 67 and 79% of the variation for 18:1, two loci explained 67 and 81% of the variation for 18:2, and seven loci explained 52 and 78% of the variation for 18:3 in these 200 F2S1 lines. The ratio of 18:1 to 18:2 was tightly interrelated as the same QTL were associated with the concentrations of 18:1 and 18:2. A quantitative trait locus that explained 63% of the phenotypic variation in the ratio of 18:1 to 18:2 is tightly linked to umc65 on chromosome 6 in the region of the linoleic acid1 locus.

The complete nucleotide sequence of apple mosaic virus RNA-3.

The complete nucleotide sequence of apple mosaic ilarvirus (ApMV) RNA-3 has been determined from cloned viral cDNAs. The 5' terminus of RNA-3 was determined by direct RNA sequencing, while the 3' end was determined by polyadenylation of genomic RNA and sub-cloning using oligo dT. ApMV RNA-3 is 2056 bases in length and encodes at least two open reading frames. It is similar in size and genome organization to the RNA-3 of other members of the Bromoviridae, which includes ilarviruses. The CP gene is in the 3' half of the molecule, and another large open reading frame is upstream of the CP gene and can potentially encode a protein of 32,400 daltons. This peptide is the same size and shows limited sequence homology to an open reading frame located at the 5' end of RNA 3 in tobacco streak and prune dwarf ilarviruses and alfalfa mosaic virus, which is postulated to be the viral movement protein. The nucleic acid sequence was not homologous to tobacco streak virus, prune dwarf virus, alfalfa mosaic virus or other members of the Bromoviridae. The 5'-non-coding region of ApMV RNA-3 contains a 15 base palindromic sequence which encloses a sequence resembling the ICR-2 regions of eukaryotic tRNA gene promoters.

The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes.

A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was flanked by 5' and 3' non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M(r) of 25,056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.