Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.
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PMID:Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase. 345 73

Dietary deficiencies in folic acid result in elevated levels of plasma homocysteine, which has been associated with the development of dementia and other neurodegenerative disorders. Previously, we have shown that elevated levels of plasma homocysteine in mice deficient for a DNA repair enzyme, uracil-DNA glycosylase (UNG), result in neurodegeneration. The goal of this study was to evaluate how deficiencies in folic acid and UNG along with elevated levels of homocysteine affect vascular cognitive impairment, via chronic hypoperfursion in an animal model. Ung(+/+) and Ung(-/-) mice were placed on either control (CD) or folic acid deficient (FADD) diets. Six weeks later, the mice either underwent implantation of microcoils around both common carotid arteries. Post-operatively, behavioral tests began at 3-weeks, angiography was measured after 5-weeks using MRI to assess vasculature and at completion of study plasma and brain tissue was collected for analysis. Learning impairments in the Morris water maze (MWM) were observed only in hypoperfused Ung(-/-) FADD mice and these mice had significantly higher plasma homocysteine concentrations. Interestingly, Ung(+/+) FADD produced significant remodeling of the basilar artery and arterial vasculature. Increased expression of GFAP was observed in the dentate gyrus of Ung(-/-) hypoperfused and FADD sham mice. Chronic hypoperfusion resulted in increased cortical MMP-9 protein levels of FADD hypoperfused mice regardless of genotypes. These results suggest that elevated levels of homocysteine only, as a result of dietary folic acid deficiency, don't lead to memory impairments and neurobiochemical changes. Rather a combination of either chronic hypoperfusion or UNG deficiency is required.
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PMID:Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil-DNA glycosylase impair learning in a mouse model of vascular cognitive impairment. 2565 13