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V M Ingram

Publications and source records attributed to V M Ingram.

At least 37 records · Page 2Linked to original sources
PubMed1985

Diversity among Purkinje cells in the monkey cerebellum.

A monoclonal antibody (B1) produced against rat embryonic forebrain membranes shows specific and striking immunohistochemical staining of Purkinje cells in the monkey cerebellum in a pattern of broad parasagittal alternating bands of cells either possessing or lacking the B1 antigen. In addition, the neurons of the deep cerebellar nuclei and some neurons of the motor cortex and of the spinal cord also contain the B1 antigen. Neurons with the B1 antigen were also seen in the somatosensory cortex, the vestibular and cochlear nuclei, and the retina.

Animals↗
PubMed1985

Growth-dependent expression of multiple species of DNA methyltransferase in murine erythroleukemia cells.

Friend murine erythroleukemia cells were found to contain three distinct species of DNA (cytosine-5-)-methyltransferase (DNA MeTase) whose relative proportions were a characteristic function of the proliferative state of the cells. Rapidly proliferating cells contained a Mr 190,000 species of DNA MeTase (DNA MeTase III), whereas cells in the late logarithmic/early plateau phase of cellular growth contained two species of Mr 150,000 and 175,000 (DNA MeTase I and II); stationary phase cells contained primarily DNA MeTase I. The three species of DNA MeTase displayed structural similarities, as determined by analysis of partial proteolysis products, and have similar de novo sequence specificities in transmethylation reactions involving purified enzyme and prokaryotic DNA. The different relative proportions of the enzymes in cells under different growth conditions suggest that the three species of DNA MeTase fulfill different roles in processes leading to the perpetuation of DNA methylation patterns.

Animals↗
PubMed1984

Differentiation of two mouse cell lines is associated with hypomethylation of their genomes.

Methyl-accepting assays and a sensitive method for labeling specific CpG sites have been used to show that the DNA of F9 embryonal carcinoma cells decreases in 5-methylcytosine content by ca. 9% during retinoic acid-induced differentiation, whereas the DNA of dimethyl sulfoxide-induced Friend murine erythroleukemia (MEL) cells loses ca. 3.8% of its methyl groups. These values correspond to the demethylation of 2.2 X 10(6) and 0.9 X 10(6) 5'-CpG-3' sites per haploid genome in differentiating F9 and MEL cells, respectively. Fluorography of DNA restriction fragments methylated in vitro and displayed on agarose gels showed that demethylation occurred throughout the genome. In uninduced F9 cells, the sequence TCGA tended to be more heavily methylated than did the sequence CCGG, whereas this tendency was reversed in MEL cells. The kinetics of in vitro DNA methylation reactions catalyzed by MEL cell DNA methyltransferase showed that substantial numbers of hemimethylated sites accumulate in the DNA of terminally differentiating F9 and MEL cells, implying that a partial loss of DNA-methylating activity may accompany terminal differentiation in these two cell types.

5-Methylcytosine↗
PubMed1983

Expression and partial DNA sequence of the chicken beta H-globin gene.

We have determined the DNA sequence of the 3' end of the putative beta H-globin gene of chickens and used an S1 nuclease protection assay to analyze transcription of this gene. Appearance of mRNA corresponding to the putative beta H-globin gene in the erythroid cells of developing chicken embryos coincides with the reported expression of the beta H-globin protein, a constitutent of hemoglobin H which is a minor component in the definitive cells of late embryos and newly hatched chickens, thereby confirming the tentative identification of this gene as beta H. Analysis of the DNA sequence of the 3'-flanking region of the beta H gene revealed the presence of a complex array of direct repeats. A part of this "repeat cluster" is homologous to an inserted element in the large intron of the goat beta A- and beta C-globin genes.

Animals↗
PubMed1983

Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.

Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.

Animals↗
PubMed1983

Globin expression in Rous sarcoma virus-transformed quail myoblasts.

Viral transformation affects certain developmental processes by blocking the differentiation of cells such as primitive muscle cells (myoblasts) or primitive red blood cells (erythroblasts). Embryonic quail myoblasts, transformed with a temperature-sensitive (ts) mutant of Rous sarcoma virus (RSV) are blocked in differentiation at the viral permissive temperature of 35 degrees C, but fuse to form myotubes at the nonpermissive temperature of 41 degrees C. We have shown that viral transformation of quail myoblasts also causes transcription of non-muscle-specific genes, ie, globins at 35 degrees C that are not observed in uninfected myoblasts or myotubes. Transformed myoblasts shifted to 41 degrees C for 5 days still contain these globin transcripts. Both alpha-like and beta-like globins are expressed.

Animals↗
PubMed1983

DNA methylation of remote sequences in chicken alpha-globin gene expression.

We have previously reported a correlation between site-specific DNA methylation and developmental expression of the chicken alpha-globin genes [8]. We have now extended this investigation to include sites 10-20 kb 5' to the alpha-globin cluster. These data indicate that the methylation status of DNA 5' to the globin genes can still be correlated with gene expression in red blood cells. The results suggest a possible role of DNA methylation in modifying regulatory sequences remote from the transcription initiation sites of globin genes; the existence of such remote regulatory sequences has also been indicated by some beta-thalassemia deletions and by genetic elements in other systems. Our results further correlate with known intermediate DNase I sensitivity in this region [7]. A discrete cluster of CpC type methylation is reported 5' to the alpha-globin gene cluster.

Animals↗
PubMed1982

Gene evolution in the chicken beta-globin cluster.

We have determined the cDNA sequence of the chicken embryonic beta-like epsilon-globin gene. Comparison with the sequences of the chicken rho-globin and beta-globin genes reveals the presence of two regions that are identical or nearly identical in epsilon and rho. The first contains the 5' untranslated sequence and exon 1, while the second region includes the second half of exon 2. Outside these regions rho and epsilon are less homologous to each other than to the adult beta-globin gene. The embryonic rho and epsilon genes are located at opposite ends of the beta-globin-gene cluster, not contiguously as are all other known pairs of simultaneously expressed globin genes. We suggest a role for gene conversion in the synchronization of expression of two highly diverged genes.

Animals↗
PubMed1982

DNA methylation in chicken alpha-globin gene expression.

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.

Animals↗
PubMed1982

Control of hemoglobin expression in chick embryonic development.

We have investigated the presence or absence of methylation of cytosine (5-MeCpG) at specific sites of the chick alpha-globin gene cluster in DNA from embryonic and adult erythroid cells, as well as from brain and sperm cells. We find that these sites are totally methylated in sperm DNA and, with some exceptions, in brain DNA. In erythroid cells no methylation or undermethylation is found in certain sites that are in or near those globin genes that are expressed in embryonic or adult cells, respectively, as predicted by the model. Other sites, away from these genes, do not necessarily follow the prediction. We have also detected a cluster of 5-MeCpC methylation sites in all tissues which is 5' to the alpha-globin cluster.

5-Methylcytosine↗
PubMed1981

cDNA sequence of a new chicken embryonic rho-globin.

In order to use specific DNA probes for the study of developmentally regulated gene expression, we have prepared cDNA clones corresponding to chicken embryonic globins by inserting cDNA.mRNA hybrids into the Pst I site of the plasmid pBR322 by using poly(dG) and poly(dC) linkers. The nucleotide sequence of the insert of one clone, representing a nearly full-length copy of an embryonic beta-like globin cDNA, has been determined. The amino acid sequence of the globin encoded by this insert is identical to the sequence of embryonic rho-globin, except for four amino acid residues near the carboxy terminus. Comparison of mRNA sequences of the embryonic and adult chicken beta-globins indicates the presence of extensive deletions in the 3' untranslated region of the embryonic gene.

Amino Acid Sequence↗
PubMed1980

In vitro transcription of chromatin containing histones hyperacetylated in vivo.

The culture of cells in the presence of sodium n-butyrate causes an accumulation of histones that are highly acetylated. When chromatin containing these histones was transcribed with E. coli RNA polymerase, an increase in the template activity compared to control chromatin was observed. Titration of chromatin with polymerase under both reinitiating and non-reinitiating conditions showed there was no increase in the number of regions available for transcription. Comparison of the kinetics for single and multiple rounds of transcription indicated that the rate of elongation was increased and probably the rate of reinitiation as well. Comparison of the size of transcripts from control and acetylated chromatin showed a small increase in the average size of transcripts from acetylated chromatin. When transcription was compared using partially purified HeLa polymerase, an increase was also seen. Studies under various ionic conditions showed that control chromatin required a higher salt concentration for optimum activity than did acetylated chromatin. In addition, at the optimum salt concentration for each chromatin, there was very little difference in the transcriptional activity using exogenous HeLa RNA polymerase.

Acetylation↗
PubMed1980

Embryonic determination and differentiation.

At the cellular level, development proceeds in a series of stages in which precursor cells are first restricted in their developmental potential (determination) and subsequently express their genetic information as specific tissues (differentiation). This paper discusses the problems encountered in seeking to understand the molecular mechanisms of thes processes and describes several model systems. A novel approach involves the inhibition of differentiation by virus transformation of the precursor cells in the chick primary mesenchyme, The transformed cells are cloned, grown to large numbers, and then analyzed biochemically.

Animals↗
PubMed1979

Isolation and transformation of primary mesenchymal cells of the chick embryo.

Pure primary mesenchymal cells from definitive streak stage chick embryos have been prepared free of epiblast and hypoblast cells. These cells have the potential in culture to differentiate into erythroid cells, beating heart muscle tissue, chondrocytes and epithelial cells. Transformation in vitro of pure primary mesenchymal cells by avian erythroblastosis virus (wt-AEV) and a temperature-sensitive mutant (ts34-AEV) gave rise to rapidly growing cells which remained largely undifferentiated, could be cloned in semi-solid medium and could be maintained for up to 3 months in culture. The majority of mesenchymal cells transformed by wt-AEV (MAE cells) are benzidine-negative. Gel electrophoresis of radioactively labeled cell proteins, immunoprecipitated with specific antisera against chicken hemoglobin, showed that MAE cell clones synthesize the alpha D, pi (or pi') and some unidentified "globin" polypeptide chains. Treatment of MAE cell clones with 1.0 mM n-butyrate stops cell proliferation reversibly and causes an increased synthesis of alpha D and pi (or pi') globin polypeptide chains. In certain clones of mesenchymal cells transformed by a temperature-sensitive mutant of the virus, ts34-AEV (MAE-ts34 cells), benzidine-positive cells can be induced by a shift from 37 degrees to 41 degrees C. The ability of the clone to undergo an increase in benzidine-positivity by temperature shift is decreased with the age of the clone. Different clones show a variable proportion of cells which are positive by immunofluorescence for both globin and chicken-specific histone H5. The alpha A and alpha D globin chains are synthesized in MAE-ts34 clones, but the ratios and quantities of these chains vary for different clones. Temperature shift made little difference in the types and quantities of globin chains synthesized; the increase in benzidine positivity is probably due to an increase in heme biosynthesis.

Alpharetrovirus↗