Search

Analysis of independently isolated clones from a mouse liver cDNA library identified a splice variant of gamma-GH mRNA with novel nucleotide sequence at the 5' end. Genomic sequencing now shows that this variant (variant II) incorporates two new alternates (exons Bla and Blb) of exon 1 in the murine gamma-GH gene remotely situated with respect to the rest of the gene. Further analysis of this variant also showed that it incorporates a small segment at the 3' end of exon A1, revealing that the previously described exon 1 consists of two individual exons (Ala and Alb) joined at a cryptic splice site. The 5' UTR and a segment of the ORF of variant II results from splicing of exon Bla to exon Blb which in turn is spliced to exon Alb and through this splicing to the rest of the exons within this gene. Remarkably, this splicing occurs even though exons Bla and Blb are located >45 Kb upstream of exons Ala and Alb. Our results also show that transcription starting at exons Bla and Blb is under the control of a separate and bidirectional promoter (promoter B). Exons Bla and Blb are located on the sense DNA strand within the complement C3 gene locus which is encoded on the antisense strand. This promoter is less efficient than the downstream promoter (promoter A) in regulating transcription at least in the context of reporter gene and primer extension assays. However, in these same contexts, this region of DNA sequence in the reverse orientation is markedly more efficient in driving transcription of an unidentified gene. Deletion of specific regions of sequence within this promoter have different effects depending upon the orientation (forward or reverse) within the reporter gene construct.

The organization and structure of the murine GH gene encoding gamma-glutamyl hydrolase were determined. The murine GH gene spans 24kb and was found to be distributed in nine exons. The intron/exon coding junctions delineated conformed to the GT-AG rule. The 5' UTR and 3' UTR along with some coding sequences were incorporated in exons 1 and 9, respectively, whereas exons 2-8 incorporated only coding sequence. A relatively GC-rich region of the genome 5' of exon 1 was distinctly promoter-like and encoded a number of putative cis-acting elements, including six Sp1 sites known to be involved in the regulation of transcription but no TATA sequence motif. Primer extension analysis of this region with mouse liver and S180 cell mRNA revealed several tsps within the region encompassing the Sp1 sites. Functional analysis of this 5' upstream region of sequence was carried out by inserting it into the pGL3 reporter gene vector for transfection into NIH3T3 cells. The transcription of the luciferase gene that resulted in these cells established the identity of this region as an active promoter for the mouse GH gene.

Mouse-liver gamma-glutamyl hydrolase (GH) is a lysosomal endopeptidase with an acid pH optimum that is activated by sulfhydryl compounds and preferentially hydrolyzes the most proximal gamma-glutamyl linkage of longer chain polyglutamates of folates and their analogues. We describe the cloning of this mouse lysosomal cDNA enzyme from liver GH mRNA in the form of two cDNA variants (1.295 and 1.268 kb in length) differing 14-fold (Variant I versus Variant II) in relative frequency that exhibited 5'-end heterogeneity and encoded alternate leader peptides. The 5' UTR in these variants also differs in length by 27 nucleotides. Otherwise, the ORF and 3' UTR in each case are the same. These cDNAs encode a protein in which the deduced amino acid sequence shares 78.9 and 69. 1% identity to rat and human GH sequences, respectively. Amino acid sequence comparisons among the three species identified three conserved Asn sites and two conserved Cys residues that may be sites of glycosylation and sulfhydryl compound activation, respectively. Variant I GH mRNA was more abundant than Variant II GH mRNA in all mouse tissues examined. Variant I GH mRNA levels were extremely high in salivary gland, moderately high in kidney, liver, lung, stomach and uterus, low in small intestine, brain and fetal liver and relatively rare in thymus, spleen and skeletal muscle. Abundance of GH mRNA among tumors varied from low to high, with no discernible correlation with their tissue of origin.