Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a novel member of the short consensus repeat family related to the carboxy terminal of the classical 150-kDa molecule.

Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.     

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense.

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Structural analysis of the three vitellogenin genes in Drosophila melanogaster.

Genomic fragments coding for sequences expressed as abundant mRNA in female Drosophila melanogaster were isolated from a lambda library. Hybridization of these clones to polytene chromosomes. in situ, identified four which mapped to X chromosomal region 9A to 9B, the locus for yolk proteins 1 and 2 (Ypl,2) and two which mapped to 12A6-7 to 12D3, the locus for Yp3. These clones were mapped with restriction enzymes, and the coding regions and regions of homology determined by Southern blots probed with cDNA, 5'-end-labelled RNA and nick-translated DNA. Heteroduplex and R-loop mapping confirmed that three of the clones carried two genes separated by about 1.4 kb and oriented in opposite directions. Southern blots probed with cDNA made from alkali-hydrolyzed RNA showed that these genes had their 5' ends next to each other. All 3 genes show homology to each other and have a main coding region of about 1.3 kb, the approximate size for the mRNAs.     

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.     

Isolation of a cDNA clone for human antithrombin III

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.     

A novel cDNA extension procedure. Isolation of chicken fatty acid synthase cDNA clones

We have developed a simple and versatile cDNA extension method using lambda-exonuclease-generated single-stranded DNA as a primer. This plasmid-based cDNA extension method can be used to synthesize unidirectional extensions of the existing cDNA clones or subcloned fragments of the untranslated and exon regions of genomic DNA clones. The method is simple to use and involves no addition of linkers or tailing. We have successfully used this method to isolate 4.6 kilobase pairs of chicken fatty acid synthase cDNA clones, starting from the fragment of a genomic clone coding for the untranslated region of the fatty acid synthase mRNA. About 2.8 kilobase pairs of the cDNA coding for the chicken fatty acid synthase has been sequenced. The sequence has an open reading frame coding for 945 amino acids of the fatty acid synthase. In the sequence, we have identified the enoyl reductase, NADPH binding region, a putative beta-ketoacyl reductase region, and the entire sequences of acyl carrier protein and the thioesterase domains. The arrangement of these partial activities in this sequence confirms the arrangement of these activities as determined through partial proteolytic mapping studies. The amino acid sequence of chicken fatty acid synthase deduced from cDNA sequences shows a high degree of homology with the rat fatty acid synthase sequence, suggesting that these multifunctional proteins are conserved evolutionarily.