Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs

When a sex hormone-binding globulin (SHBG) cDNA was used to screen a human testicular cDNA library, three distinct cDNAs were isolated, one of which corresponds to the human SHBG cDNA sequence and probably represents testicular androgen-binding protein. The other two SHBG-related cDNAs each contain unique 5' regions that diverge from the SHBG cDNA sequence at the same position, and one of them (SHBGr-2) lacks a 208-base pair region within the SHBG cDNA. As a result, this cDNA could potentially encode for a truncated form of SHBG which lacks N-linked carbohydrates and part of the steroid-binding domain. Southern blots of human placental DNA and cloned genomic DNA fragments also indicate that SHBG and its related testicular cDNAs are the products of a single gene. Sequence analysis of the gene indicates that the complete coding region for the SHBG precursor is comprised of 8 exons, which are distributed over 3.2 kilobase (kb) of genomic DNA, and the unique 5' regions associated with the two SHBG-related testicular cDNAs were identified 1.9 kb upstream from the initiating codon for SHBG. In addition, the deletion within SHBGr-2 is due to the removal of exon 7, and an interesting feature of the gene is that differentially used exons are preceded by Alu repetitive DNA sequences. Although the relative abundance of the various SHBG-related mRNAs in the testis has not been established, Northern blot analysis indicates that they are similar in size (1.6 kb) to that of hepatic SHBG mRNA.     

Structural characterization of exon 6 of the rat IGF-I gene.

In rat liver, insulin-like growth factor I (IGF-I) mRNAs exist as two major size classes of 7.5-7.0 kb and 1.2-0.9 kb. The 7.5- to 7.0-kb IGF-I mRNAs predominate in some nonhepatic tissues of the rat. Because the previously reported sequences of rat IGF-I cDNAs and genomic clones account for only 2.1 kb of sequence, the majority of the sequence of 7.5- to 7.0-kb rat IGF-I mRNAs was unknown. Using a combination of nucleotide sequencing of genomic DNA and cDNA clones and Northern hybridization and RNase protection, we have characterized a 6,354-base-long 3' exon (exon 6) of the rat IGF-I gene. The sequence of exon 6 establishes the previously unknown sequence of the 3' end of the 7.5- to 7.0-kb rat IGF-I mRNAs, comprised predominantly of an unusually long 3' untranslated sequence (3'UT). The long 3'UT contains multiple ATTTA, A(T)nA, and (T)nA sequences, as well as inverted repeats. These sequences may contribute to the shorter half-life of the 7.5- to 7.0-kb rat IGF-I mRNAs relative to the 1.2- to 0.9-kb forms that have been demonstrated previously in vitro and in vivo. We also demonstrate that the 7.5- to 7.0-kb rat IGF-I mRNAs are localized to the cytoplasm of rat liver, providing indirect evidence that they are mature and functional mRNAs.     

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.