Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase

Abstract

A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

Cloning, Expression, Activity and Folding Studies of Serine Hydroxymethyltransferase: A Target Enzyme for Cancer Chemotherapy

“…The active form of this enzyme is widely distributed in nature, including both prokaryotic and eukaryotic organisms. The gene encoding cytoplasmic SHMT is located on human chromosome 17p11.2, whereas the gene for mitochondrial SHMT is found on chromosome 12q13.2 [Byrne et al, 1992;Garrow et al, 1993]. A similar gene sequence is also found on human chromosome 1p32.3-p33, which has been identified as processed pseudogene SHMT-ps1 [Byrne et al, 1996;Devor et al, 1997].…”

“…The two isoforms of SHMT are found in eukaryotic cells; one expressed in the cytoplasm and the other in mitochondria. Human cells have also been reported to possess genes for the two isoforms [Byrne et al, 1992;Garrow et al, 1993]. A third member of this small gene family has also been recognized as a pseudogene, SHMT-ps1, located on human chromosome 1p32.3-p33 [Byrne et al, 1996;Devor et al, 1997].…”

Effects of Breed, Parity, and Folic Acid Supplement on the Expression of Folate Metabolism Genes in Endometrial and Embryonic Tissues from Sows in Early Pregnancy1

“…Similarity analysis showed that the PCR product for porcine cSHMT is 88% identical to the sheep [17], human [25], and rabbit [26] nucleotide sequences. The deduced amino acid sequence showed identities of 96%, 95%, and 94% between porcine cSHMT and cSHMT from sheep [17], human [25], and rabbit [26], respectively. Analysis of the T-protein nucleotide sequence (fragment a; Fig.…”

A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design

“…The degenerate oligonucleotide-primed PCR (DOP-PCR) has been applied to the characterization of abnormal chromosomes and also in the cloning of new markers for specific chromosome regions [ 13 ]. Based on unknown genome information circumstances, using DOP-PCR to obtain glyA (encoding SHMT) can overcome the shortcomings of existing methods, such as cDNA library [ 14 , 15 ], southern-blot hybridization [ 16 ], direct PCR amplification according to the known genome information [ 17 , 18 ], and shotgun technology [ 19 ], for their waste time and material under the same conditions. Protein engineers redesign proteins in order to improve their biomedical or industrial utility [ 20 ].…”