The discovery of new intron-containing human tRNA genes using the polymerase chain reaction

Introns in transfer RNA genes are rare in vertebrates. Until now, the only intron-containing human tRNA genes were believed to be those coding for tRNA(Tyr). All of these introns are inserted 3' to the anticodon position in these genes. We have designed polymerase chain reaction primers that can amplify all of the tRNA(Tyr) genes for cloning and sequencing by using the conserved portions of the gene coding for the structural part of the tRNA. Our preliminary results have revealed five tRNA(Tyr) genes, each of which contains a different intron. We used the same technique to amplify, clone, and sequence the human genes for tRNA(Leu)CAA. This has resulted in the discovery that this human tRNA gene family also has introns inserted 3' to the anticodon. This polymerase chain reaction technique is useful in detecting new families of intron-containing tRNA genes as well as identifying sequence variations in the introns of individual genes.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.     

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14.

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts coding for the entire gene. The genomic DNA was isolated and studied by restriction mapping, polymerase chain reaction analysis, and DNA sequencing. The gene was 11.5 kilobases in length and consisted of five exons separated by four introns. In addition, 0.8 kilobases of DNA from the 5'-flanking region were sequenced. The exon-intron boundaries all observed the "GT-AG" rule. The gene for protein C inhibitor was assigned to chromosome 14 by polymerase chain reaction analysis of human/hamster hybrid cell lines. The organization of the gene for protein C inhibitor is similar to the genes coding for alpha 1-antitrypsin and alpha 1-antichymotrypsin. The genes for these two proteins are also localized on chromosome 14 suggesting a recent evolution of the genes for these three proteins from a common ancestor.     

Genotyping of fimbrial adhesins in<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from slovak piglets suffering from diarrhea

One-hundred sixty Escherichia coli isolates obtained from piglets with diarrhea from different parts of Slovakia were examined for the presence of genes coding for F4, F5, F6 and F41 fimbrial adhesins, and hemolytic activity. According to polymerase chain reaction tests 74 (46%) E. coli isolates were positive for primers that detected genes coding for fimbrial adhesins. Of these 74 isolates, 64 were positive for genes encoding for F4+, four for F5+, five for F6+, and one for both F41+ and F5+ adhesins.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene

The utilization of micro-algae for the production of food and fine chemicals is growing in importance. However, confusion of names and species makes comparison of results by different authors very difficult. In this work, five species of Dunaliella were characterized using their 18S ribosomal RNA genes. Conserved oligonucleotides complementary to 5' and 3' termini of the 18S rDNAs were designed and utilized to amplify theme, restriction fragment length polymorphism analysis of the polymerase chain reaction products was developed. Species-specific primers were also designed and utilized to corroborate the identification.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.     

Inference of positive and negative selection on the 5' regulatory regions of Drosophila genes

Both positive selection and negative selection have been shown to drive the evolution of coding regions. It is of interest to know if the corresponding 5' regions of genes may be subjected to selection of comparable intensities. For such a comparison, we chose the Accessory gland protein (Acp) genes as our test case. About 700 bp and 600 bp for the 5' and coding regions, respectively, of eight previously unstudied genes were sequenced from 21 isogenic lines of D. melanogaster and one line from D. simulans. The ratio of divergence at the amino-acid replacement sites (A) over that at the synonymous sites (S) was twice the ratio for common polymorphism. Interestingly, the 5' region shows the same trend, with the 5'/S divergence ratio being 1.8 times higher than the 5'/S ratio for common polymorphism. There are several possible explanations for the 5'/S ratios, including demography, negative selection, and positive selection. Under normal conditions, positive selection is the most likely explanation. If that is true, about 45 to 50 percent of all fixed differences at both the replacement and 5' sites were adaptive, even though the substitution rate in the former is only half that of the latter (K(A)/K(S) approximately 0.3 vs. K(5')/K(S) approximately 0.6). As previous analyses have indicated, the inclusion of slightly deleterious polymorphism confounds the inference of positive selection. The analysis of published polymorphism data covering 97 verified 5' regions of Drosophila suggests more pronounced selective constraint on the 5' untranslated region and the core promoter (together corresponding to approximately 200 bp in this data set) when compared to the more distal portion of the 5' region of genes.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.     

The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions

Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.     

Screening for mutations in the promoter and the coding region of the IGFBP1 and IGFBP3 genes in Silver-Russell syndrome patients.

In the present study we sought to identify genetic variation in genes for insulin-like growth factor binding proteins 1 and 3 (IGFBP1, IGFBP3) in 7p12-13 which through alteration of protein function or level of expression might contribute to the manifestation of Silver-Russell syndrome. Genomic DNA samples from 49 Silver-Russell syndrome (SRS) patients and from unaffected controls were investigated by single-strand conformation analysis. Overlapping polymerase chain reaction fragments covered the whole coding sequences as well as the 5' untranslated region of the IGFBP1 and IGFBP3 genes. We detected 3 new polymorphisms in the transcribed sequence of IGFBP1, one amino acid polymorphism in exon 1 of IGFBP3 and four variants in its promotor region and in intron 1. They all occurred in similar frequencies in SRS patients and in controls. Thus, paternally inherited mutations in the promoter and coding regions of IGFBP1 and IGFBP3 genes play neither a major nor a minor role in the etiology of SRS. The newly detected polymorphisms in the coding region are powerful tools for analysis of imprinting status and for detection of possible changes in the imprinting patterns of the two genes.