Growth Phase-Dependent Regulation of Vsr Endonuclease May Contribute to 5-Methylcytosine Mutational Hot Spots in Escherichia coli

Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine methylase of Escherichia coli is maintained at a constant level during cell growth, while Vsr endonuclease levels are growth phase dependent. Decreased production of Vsr relative to Dcm during the log phase may contribute substantially to the mutability of 5-methylcytosine.     

Effects of Amino Acid Substitutions at the Active Site in Escherichia Coli β-Galactosidase

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme β-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac(-). Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac(+). The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.     

Maternal factors in onset of Huntington disease.

Analyses of father-offspring and mother-offspring similarity in onset age suggest that nuclear genes account for a significant portion of the modification of onset age in Huntington disease. The effects of non-nuclear modifiers are supported by the finding that the offspring of affected women have significantly older mean ages of onset than offspring of affected men irrespective of the onset age in the parent. The absence of increased father-daughter similarity indicates that modification is not X-linked. The absence of reproductive advantage for late-onset individuals and the absence of a multigenerational maternal-lineage effect suggest that the modifying effect of the sex of the affected parent occurs in a single parental generation. Offspring of affected women with onset between ages 35 and 49 had a significantly older mean onset age than their mothers. This suggests that a protective effect may be conferred upon the offspring of affected women.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Inverse relationship between age at onset of Huntington disease and paternal age suggests involvement of genetic imprinting.

It is well recognized that age at onset of Huntington disease (HD) is strongly influenced by the sex of the affected parent, and this has lead to suggestions that genetic imprinting or maternal specific factors may play a role in the expression of the disease. This study evaluated maternal and paternal ages, birth order, parental age at onset, and sex of the affected parent and grandparent in 1,764 patients in the National HD Roster by using linear-regression techniques which incorporated a weighted least-squares approach to accommodate the correlation among siblings. It was found that paternal age is negatively associated with age at onset of HD, particularly among subjects who inherit the mutant gene from grandfathers. Apparent associations between age at onset and birth order and between age at onset and maternal age were not significant after adjustment for paternal age. The paternal age effect is strongest among juvenile-onset cases and individuals with anticipation of greater than or equal to 10 years, although it is detectable across the entire age-at-onset distribution. The tendency for older fathers, including those not transmitting the HD gene, to have affected offspring with early-onset disease may be consistent with a gene imprinting mechanism involving DNA methylation. Because paternal age in unaffected fathers is also a significant determinant of age at onset, methylation in this context might involve HD modifier genes or the normal HD allele.     

A Familial Factor Independent of CAG Repeat Length Influences Age at Onset of Machado-Joseph Disease

Machado-Joseph disease (MJD) is a late-onset, progressive, neurodegenerative disorder caused by the expansion of an unstable trinucleotide (CAG) repeat sequence in a novel gene (MJD1) on chromosome 14. Previous studies showed that age at onset is negatively correlated with the number of CAG repeat units, but only part of the variation in onset age is explained by CAG repeat length. Ages at onset and CAG repeat lengths of 136 MJD patients from 23 kindreds of Portuguese descent were analyzed, to determine whether familial factors independent of CAG repeat length modulate age at onset of MJD. Correlation among sibs for onset age adjusted for CAG repeat length was .43, which indicates that an environmental or genetic factor common to sibs influences onset age. Positive correlations were also observed for avuncular (r = .22) and first-cousin pairs (r = .28), which supports the hypothesis that a genetic factor is influencing age at onset. Commingling analysis of onset ages adjusted for CAG repeat length identified three distributions in this population of affected individuals. Further studies of a much larger sample are needed to determine whether these distributions represent the influence of a genetic or environmental factor.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.