RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Centrifugation of 2-cell mouse ova: cytoplasm stratification and recovery

Summary Two-cell mouse ova, which were centrifuged for l h at 70 000–90 000xg, showed a precise stratification of the cytoplasm and an elongation of the nucleus. The ova were fixed at different times and observed by light and electron microscopy using cytochemical methods and detergent extractions. Within 40 min after centrifugation the normal-looking morphology was recovered except for the persisting lipid caps at the centripetal poles of the blastomeres. Cleavage, compaction and blastulation were not prevented by centrifugation. Treatments with colcemid or cytochalasin D delayed but did not impair recovery. These results suggest that a resilient cytoskeletal structure may be involved in this kind of embryonic regulation.     

Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA.

5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA.     

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Steady state terrestrial ecosystems and the global carbon cycle

The assumption that landscapes dominated by mature vegetation are presently in carbon steady state with the atmosphere is challenged. Evidence suggests that the vegetation and soils of these landscapes are frequently disturbed and over short time periods (<300 yr) slowly sequester atmospheric carbon. The critical consideration in this argument is the time interval used to evaluate a steady state. Current models of carbon flux through the terrestrial biota limit their time considerations to 120 yr, a short and inadequate time interval for realistic assumptions about steady state in the carbon cycle of vegetation.Research performed under subcontract 19B-07762C with S. Brown and 19X-43326C with the Center for Energy and Environment Research of the University of Puerto Rico (A. E. Lugo) under Martin Marietta Energy Systems, Inc., contract DE-AC05-840R21400 with the U.S. Department of Energy.     

Long poly(A) tracts in the human genome are associated with the Alu family of repeated elements

Long poly(dA).poly(dT) tracts (poly(A) tracts), regions of DNA containing at least 20 contiguous dA residues on one strand and dT residues on the complementary strand, are found in about 2 X 10(4) copies interspersed throughout the human genome. Using poly(dA).poly(dA) as a hybridization probe, we identified recombinant lambda phage that contained inserts of human DNA with poly(A) tracts. Three such tracts have been characterized by restriction mapping and sequence analysis. One major poly(A) tract is present within each insert and is composed of from 28 to 35 A residues. In each case, the poly(A) tract directly abuts the 3' end of the human Alu element, indicating that the major class of poly(A) tracts in the human genome is associated with this family of repeats. The poly(A) tracts are also adjacent to A-rich sequences and, in one case, to a polypurine tract, having the structure GA3-GA3-GA4-GA6-GA5-GA4. We suggest that repetitive cycles of unequal crossing over may give rise to both the long poly(A) and polypurine tracts observed in this study.