Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.     

Cloning, expression and sequence homologies of cDNA for human gamma enolase

The nucleotide sequence of the human gamma-enolase mRNA was determined from recombinant cDNA clones. The sequence spans 2273 bp and includes the complete coding region of 1299 bp, a 5'-noncoding region of 74 bp and a 897-bp-long 3'-noncoding region containing a variant polyadenylation signal (ATTAAA). The deduced amino acid (aa) sequence is 433 aa long and shows a 97% similarity with rat gamma-enolase. Both the 5'- and 3'-untranslated regions are similar (82% and 68%, respectively) to the analogous regions of the rat gamma-enolase gene, suggesting that a strong selective pressure operates on noncoding segments of gamma-enolase mRNAs. The size of the gamma-enolase mRNA expressed in human brain is 2.4 kb. A crosshybridizing 1.5-kb message is detected in human skeletal muscle which may be derived from the beta-enolase-coding gene.     

Molecular cloning, expression, and chromosomal localization of a ubiquitously expressed human 6-phosphofructo-2-kinase/ fructose-2, 6-bisphosphatase gene (PFKFB3)

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15-->p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.