Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an 'uncloneable' phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this 'uncloneable' fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNAThr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNAThr. These results imply that the HeLa mitochondrial tRNAThr gene is responsible for the 'uncloneable' phenotype of this region of human mitochondrial (mt) DNA. A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNAThr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T----G:C transitions. A preference for A:T----G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

Two Circular Single-stranded DNAs Associated with Banana Bunchy Top Virus

Nucleic acids extracted from partially purified banana bunchy top virus (BBTV) consisted of 20 Kb DNA, 0.9-1.1 Kb DNA and 0.3 Kb RNA. Partially purified BBTV preparations predigested with DNase and RNase before particle disruption and nucleic acid isolation yielded only the 0.9-1.1 Kb DNA, but no corresponding nucleic acid band was obtained in total nucleic acid isolated from healthy banana tissue. Analysis of two BBTV cDNA clones showed that clone 1 consisted of 287 nucleotides and clone 2 contained a 1.0 Kb DNA insert. Clone 1 is not part of clone 2. When two pairs of primers, each pair in opposite orientation were used to amplify BBTV DNA by PCR using the total DNA from diseased banana tissues or DNA encapsidated in BBTV particle as the template, a DNA product of 1.1 Kb was generated by both, results indicating that the BBTV DNAs are circular. Additional results suggested that BBTV contained at least two circular ssDNAs designated BBTV essDNA I (containing clone 1 nucleotide sequence) and BBTV, cssDNA II (containing clone 2 nucleotide sequence).     

Two Circular Single-stranded DNAs Associated with Banana Bunchy Top Virus

Nucleic acids extracted from partially purified banana bunchy top virus (BBTV) consisted of 20 Kb DNA, 0.9–1.1 Kb DNA and 0.3 Kb RNA. Partially purified BBTV preparations predigested with DNase and RNase before particle disruption and nucleic acid isolation yielded only the 0.9–1.1 Kb DNA, but no corresponding nucleic acid band was obtained in total nucleic acid isolated from healthy banana tissue. Analysis of two BBTV cDNA clones showed that clone 1 consisted of 287 nucleotides and clone 2 contained a 1.0 Kb DNA insert. Clone 1 is not part of clone 2. When two pairs of primers, each pair in opposite orientation were used to amplify BBTV DNA by PCR using the total DNA from diseased banana tissues or DNA encapsidated in BBTV particle as the template, a DNA product of 1.1 Kb was generated by both, results indicating that the BBTV DNAs are circular. Additional results suggested that BBTV contained at least two circular ssDNAs designated BBTV cssDNA I (containing clone 1 nucleotide sequence) and BBTV cssDNA II (containing clone 2 nucleotide sequence).     

Sequence of a HeLa cDNA provides the DNA binding domain and carboxy terminus of HE47: a human helix-loop-helix protein related to the enhancer binding factor E47.

In order to identify cDNA encoding regulators of gene expression, a HeLa lambda gt11 expression library was screened with a DNA segment containing multiple copies of a sequence spanning the AP4 site in the simian virus 40 control region. We identified a partial cDNA encoding HE47, a sequence-specific DNA binding protein. The HeLa clone overlaps with a previously reported human B-cell partial cDNA encoding E47. The HeLa cDNA includes the HE47 DNA binding domain, its carboxy terminus, and the 3' untranslated region of its mRNA.     

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.     

Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an ‘uncloneable’ phenotype

Several regions of the human mitochondrial genome are refractory to cloning in plasmid and bacteriophage DNA vectors. For example, recovery of recombinant M13 clones containing a 462 basepair MboI-Kpn I restriction fragment that spans nucleotide positions 15591 to 16053 of HeLa cell mitochondrial DNA was as much as 100-fold lower than the recovery of M13 clones containing other regions of the human mitochondrial genome. All of 50 recombinant M13 clones containing this ‘uncloneable’ fragment had one or more changes in nucleotide sequence. Each clone contained at least one alteration in two nucleotide positions within the tRNA^Thr gene that encode portions of the anticodon loop and D-stem of the HeLa mitochondrial tRNA^Thr. These results imply that the HeLa mitochondrial tRNA^Thr gene is responsible for the ‘uncloneable’ phenotype of this region of human mitochondrial (mt) DNA.A total of 61 nucleotide sequence alterations were identified in 50 independent clones containing the HeLa mt tRNA^Thr gene. 56 mutations were single-base substitutions; 5 were deletions. Approximately 80% of the base substitution mutations were A:T → G:C transitions. A preference for A:T → G:C transition mutations also characterizes polymorphic base substitution variants in the mitochondrial DNA of unrelated individuals. This similarity suggests that human mitochondrial DNA sequence variation within and between individuals may have a common origin.     

A low-copy-number Sorghum DNA sequence that detects hypervariable EcoRV fragments

A sorghum genomic DNA clone that hybridized on Southern blots in simple but different patterns to fragments produced by digestion of DNA from the parents of an F₂ mapping population was hybridized to EcoRV-digested DNA from 53 accessions. Forty-six different fragment patterns were observed, each comprised of from one to ten bands. Much less variability was detected in EcoRI than EcoRV digests of a selected subset of the accessions. Base-sequence analysis of the clone did not reveal a functional identity for the sequence and the clone does not contain repeated sequences often associated with hypervariable loci. Clones such as this will be especially useful in evaluating germplasm diversity and in identifying the potential parentage of hybrids.     

Structure of circular copies of the 412 transposable element present in Drosophila melanogaster tissue culture cells, and isolation of a free 412 long terminal repeat

We have isolated, from Drosophila melanogaster tissue culture cells, extrachromosomal circular forms of the transposable element 412, and have cloned some of them in bacteriophage lambda. A total of 24 clones have been analysed in detail by restriction and heteroduplex mapping. Seventeen clones are virtually identical, and contain complete 412 elements with one copy of the long terminal direct repeat (LTR). The remaining seven clones are all different and contain various rearrangements. Four have deletions, two have some 412 sequence substituted by other DNA and one has both an inversion and a deletion. The clone containing the inversion has two LTRs in inverted orientation and separated by a few thousand bases of 412 DNA. The base sequences of the two LTRs in this clone, and of the LTR in one of the 17 clones containing complete elements are very similar to that of the 481 base-pair LTR of a genomic 412 element. We have found no evidence, in either cloned or uncloned material, for 412 elements with two LTRs as a tandem direct repeat. We have found that there are several "free" 412 LTRs in genomic DNA from D. melanogaster strains Canton S and Oregon R, and from D. melanogaster tissue culture cells. We have cloned and sequenced one of these free LTRs. It is 475 base-pairs long and is flanked by a direct repeat four base-pairs long. This sequence differs from that of the 481 base-pair repeat at 16 places including a ten base deletion.     

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.     

Molecular cloning and functional characterization of a cDNA encoding nucleosome assembly protein 1 (NAP-1) from soybean

NAP-1, a protein first isolated from mammalian cells, can introduce supercoils into relaxed circular DNA in the presence of purified core histones. Based on its in vitro activity, it has been suggested that NAP-1 may be involved in nucleosome assembly in vivo. We isolated a cDNA clone encoding a soybean NAP-1 homolog, SNAP-1. The SNAP-1 cDNA contains an open reading frame of 358 amino acid residues with a calculated molecular weight of 41 kDa. The deduced amino acid sequence of SNAP-1 shares sequence similarity with yeast NAP-1 (38%) and human hNRP (32%). Notable features of the deduced sequence are two extended acidic regions thought to be involved in histone binding. SNAP-1 expressed in Escherichia coli induces supercoiling in relaxed circular DNA, suggesting that SNAP-1 may have nucleosome assembly activity. The specific activity of SNAP-1 is comparable to that of HeLa NAP-1 in an in vitro assay. Western analysis reveals that SNAP-1 is expressed in the immature and young tissues that were examined, while mature tissues such as old leaves and roots, show very little or no expression. NAP-1 homologs also appear to be present in other plant species.     

Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element

An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence.     

Sequence analyses of extrachromosomal Sau3A and related family DNA: analysis of recombination in the excision event.

Previously, we reported a recombination-prone human alphoid-like repetitive DNA (Sau3A family) which is characterized by abundance in the extrachromosomal fraction and restriction fragment length polymorphism. We suggested a specific homologous recombination to be responsible for the DNA excision from the chromosomes and also the sequence rearrangement in the chromosomes. In order to investigate the nature of the recombination further, 8 different clones were obtained which hybridized with Sau3A probe among over 1,500 extrachromosomal DNA clones. Restriction mapping and nucleotide sequence analyses showed two to be Sau3A monomers and dimers, four Sau3A recombinants, as observed previously, one a recombinant of the Sau3A-related sequence on chromosome 17, and one a new Sau3A-related sequence. Sequence analyses of the recombination junctions in the recombinant clones indicated a specific homologous recombination also to be responsible for all but one clone. The molecular mechanism and biological significance associated with the recombination are discussed.     

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.     

Mapping using unique sequences

Theoretical predictions are given for the progress expected, when mapping DNA by identifying clones containing specific unique sequences. Progress is measured in three ways; however, all results depend on (dimensionless counterparts of) the number of clones and the number of unique sequences used. Furthermore, the effects of clone length dispersion are included in the theoretical predictions. Both the clones in the library and the unique sequences are assumed to be generated randomly, with uniform probability of originating at any base in the region to be mapped. The first measure of progress is the expected length fraction of the region to be mapped covered by at least one clone, when clones containing at least one unique sequence are included in the map. The second measure of progress is the expected length fraction of the region to be mapped in "covered intervals", an interval being the region between adjacent unique sequences. Alternative definitions for clones covering an interval are analyzed. The third measure of progress is the expected number of clone islands generated; an island covers successive intervals. Finally, using these measures of progress, we compare the efficiency of this new mapping strategy with conventional clone mapping strategies.     

Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.     

The ILtat 1.4 surface antigen gene family of Trypanosoma brucei.

The cDNA sequence for the variable surface glycoprotein (VSG) expressed in Trypanosoma brucei clone ILtat 1.4 (called clone D for brevity) hybridizes strongly to three regions in trypanosome genomic DNA. These three regions were extensively characterized by Southern hybridization analyses, genomic DNA cloning and DNA sequence determinations. All three regions occur in the genomes of all trypanosome clones of the ILTAR 1 repertoire regardless of whether or not VSG D was being expressed. Extensive (clone dependent) DNA rearrangements and a (clone independent) double strand DNA break were found distal to the 3'-end of the VSG D coding sequence of one of the regions. VSG D mRNA is most likely synthesized from this region, but a recombinant DNA clone of the VSG coding sequence could not be obtained for confirmation. Recombinant clones of the other two regions were obtained. DNA sequence analyses revealed that their coding sequences differ from each other by 17%. They differ from the ILtat 1.4 cDNA sequence by 4% in one case, and 13% in the other. By analogy with another VSG gene system, one of these two regions may have originally given rise to the third region from which the mRNA is probably transcribed.