DNA methylation and cell memory

In this paper we address the question: How do replicating mammalian cells remember with high fidelity their proper state of differentiation? Several possible mechanisms for cell memory are discussed, and it is concluded that only mechanisms involving DNA methylation are supported by strong experimental evidence. This evidence is reviewed. The establishment and modulation of methylation patterns are discussed and a hemimethylation model for stem cells is presented. The overall conclusion is that, although little is yet known about the details, there should be little doubt about the existence of a methylation system functioning at least to aid cell memory.     

Biological activity of the mammalian RAP genes in yeast.

We have screened expression libraries for mammalian cDNAs capable of suppressing defects in ras1- Schizosaccharomyces pombe. Both the RAP1A and RAP1B genes were identified in this manner. They suppress defects in cell morphology and sporulation, although not conjugation. In contrast, RAP genes do not suppress phenotypes in the yeast Saccharomyces cerevisiae that are deficient in RAS. Indeed, expression of RAP1A appears to antagonize the activated S. cerevisiae RAS2val19 gene. These results indicate that RAP proteins can interact with RAS targets, sometimes productively, sometimes nonproductively.     

Oxygen binding and aggregation of bullfrog hemoglobin

The hemoglobin of the bullfrog, Rana catesbeiana, forms aggregates larger than tetramers in two ways. The first, which results from intermolecular disulfide bonds, can be prevented by treatment with iodoacetamide. The second way results from the association of the deoxygenated forms of the two major components, B and C, to form reversibly an aggregate which is believed to be a trimer, BC2. The sedimentation velocity data show that the stoichiometry of the aggregate cannot be 1:1. The electrophoretic pattern of the deoxygenated B/C mixture suggests that the association is not indefinite. No significant aggregation of the separate deoxygenated tetramers of the components nor of the oxygenated components or mixture occurs. Gel chromatography of the oxygenated forms of components B and C and of mixtures indicates that the B and C tetramers both form dimers upon dilution with a dissociation constant of 2-3 micron. The oxygen-binding data indicate that the B/C aggregate has a much lower oxygen affinity than its constituent tetramers. Dissociation of the low affinity B/C aggregate to higher affinity B and C tetramers with increasing oxygenation gives rise to enhanced cooperativity as measured by the Hill coefficient which is maximal near 75-80% oxygenation and is as high as 4.1 at a heme concentration of 15 mM.     

Syntheses of Phosphonate Analogues of Dideoxyadenosine (DDA)-, Dideoxycytidine (DDC)-, Dideoxyinosine (DDI)-, and Deoxythymidine (DDT)-5′-Monophosphates

Abstract

Phosphonate derivatives of ddA, ddC, ddI and ddT (5f, 5e, 5c, and 5a) were prepared by condensing the 5′-aldehydes with diphenyl triphenylphosphoranylidenemethylphosphonate, reducing the resultant olefins and hydrolyzing the phosphonate phenyl esters, sequentially, with base and then C. atrox phosphodiesterase.     

Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies

An important consideration in interpreting indices of gene expression in human bone is relating mRNA levels to functional endpoints such as bone architecture. In the present study, a method was developed for quantitative measurement of gene expression and bone morphology in the same specimen. Three-dimensional images of iliac crest bone biopsies from healthy premenopausal women were obtained using a novel high resolution cryogenic mu-CT scanner. RNA was isolated from the biopsies and mRNA levels were measured for genes related to bone metabolism. The gene expression profile and variability of expression within iliac crest biopsies of women was similar to human osteoblastic cell lines and rat long bones. mRNA for alkaline phosphatase, bone matrix proteins, and selected cytokines and cytokine receptors were consistently detected in biopsies. As previously shown in rat bone, there was a tight correlation between mRNA levels for type 1 collagen and osteonectin, a weaker correlation between type 1 collagen and osteocalcin and no correlation between bone matrix proteins and alkaline phosphatase. The relative abundance of the mRNA for the three most prevalent transforming growth factor-beta (TGF-beta) isoforms in bone (TGF-beta(1)> TGF-beta(3)> TGF-beta(2)) was the same as the known abundance of the corresponding TGF-beta peptides in bone matrix. The results demonstrate the feasibility of analyzing the three-dimensional architecture of a bone biopsy using cryogenic mu-CT imaging and then measuring expression of genes related to bone cell function within the same specimen following RNA extraction and analysis.     

A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe.

Byr3 was selected as a multicopy suppressor of the sporulation defects of diploid Schizosaccharomyces pombe cells that lack ras1. Like cells mutant at byr1 and byr2, two genes that encode putative protein kinases and that in multiple copies are also suppressors of the sporulation defects of ras1 null diploid cells, cells mutant at byr3 are viable but defective in conjugation. Nucleic acid sequence indicates byr3 has the capacity to encode a protein with seven zinc finger binding domains, similar in structure to the cellular nucleic acid binding protein (CNBP), a human protein that was identified on the basis of its ability to bind DNA. Expression of CNBP in yeast can partially suppress conjugation defects of cells lacking byr3.