Identification and characterization of two new human mu opioid receptor splice variants,hMOR-1O and hMOR-1X

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.     

Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

A novel H-2K splice form: predictions for other alternative H-2 splicing events

A large number of H-2K and H-2D cDNA clones from a C3HfB/HeN spleen cDNA library were extensively characterized. All H-2D^k cDNAs were shown to exhibit the short form of exon 8, consistent with the presence of a single lariat branchpoint site within intron 7. Twenty-five H-2K^km2 cDNAs were found to bear a short exon 8, whereas only two clones were shown to carry the longer form of this exon. In one of the H-2K^km2 cDNAs, a novel pattern of H-2 splicing was identified, in which an extra 15 nucleotides, derived from the 3′ end of intron 5, were inserted between the intact and unaltered exon 5 and exon 6 sequences. Resulting from the apparent use of a cryptic splice acceptor site in place of the canonical intron 5 site, this insertion is predicted to generate an in-frame insertion of five nonpolar amino acid residues within a highly polar region of the intracytoplasmic domain of the H-2K polypeptide. The features of this novel splice form served as the basis for predicting additional rare, alternative H-2 pre-mRNA splicing events that might produce functionally relevant microheterogeneity in the encoded H-2 gene products.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.