Sequence Rearrangements Research Articles

The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism

Abelson murine leukemia virus-transformed precursor B lymphocytes from sci (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their lg heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and J H locus to from aberrant “DJ H” rearrangements and secondary rearrangement of sequences from within the V H locus to the aberrant “DJ H” intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamer-nonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments.

Insertion sequence IS2 in the cyanobacterium Chlorogloeopsis fritschii

A cloned DNA fragment, previously demonstrated to encode ribulose bisphosphate carboxylase/oxygenase (RuBisCO) of Chlorogloeopsis fritschii strain CCAP1411/1b, is shown also to include the entire transposable element, IS2, normally a resident in the Escherichia coli genome. Southem-blot hybridisation experiments confirm the presence of IS2 in the C. fritschii genome. This finding adds a new and unrelated species to the known host range of this element and provides evidence of genetic transfer between the Gram-negative E. coli and cyanobacteria. This may also have significance in relation to the nucleotide sequence rearrangements known to occur adjacent to RuBisCO and nif genes in other nitrogen-fixing cyanobacteria.

Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: Evidence for an increased frequency of deletions/additions with aging

To obtain information on the extent of random nucleotide changes in mitochondrial DNA (mtDNA) from different organs of young adult and senescent Fischer 344 rats, the temperature of thermal denaturation ( t m ) was measured in (1) the native mtDNA cut at a single SstI site and (2) the reannealed duplexes formed after the initial melting of the mtDNA sample. No change was found between the two t m values in either young or senescent mtDNA, suggesting that the overall mismatch in nucleotide sequence in these samples was below the resolution of the method estimated at about 0.2%. In another experiment, mtDNA samples from young adult or senescent BALB/c mouse liver were digested with EcoRI, denatured and allowed to reanneal. The duplexes formed by the 14-kb EcoRI fragment were analyzed in randomly taken electron micrographs for the occurrence of mismatched segments. About 1.8% of reconstituted duplexes in adult mtDNA and 11% of those in senescent mtDNA contained small loops or knobs suggestive of deletions/additions of about 400 ± 150 nucleotides. These data correspond to about 1% of the native mtDNA population in adult liver and about 5% in senescent liver having deleted/inserted segments. Although deletions/insertions may occur at variable sites, their distribution appears to be non-random. These findings suggest that small sequence rearrangements, which have been observed previously in unicircular dimers of mouse and human mtDNA, occur also in monomeric mtDNA from normal tissues and accumulate with aging.

Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells

A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV- gpt and HSV tk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSV tk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV- gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSV tk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV- gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed ‘sectoring’ on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV- gpt gene in these cells were isolated while maintaining selection for the HSV tk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.

Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells

A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV- gpt and HSV tk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSV tk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV- gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSV tk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damage SV- gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed ‘sectoring’ on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV- gpt gene in these cells were isolated while maintaining selection for the HSV tk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induce mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.

The NADH-dehydrogenase subunit 5 gene in Oenothera mitochondria contains two introns and is co-transcribed with the 5 S rRNA gene

The gene encoding subunit 5 of the NADH dehydrogenase complex (ND 5) has been identified in Oenothera mitochondria from a cDNA clone. The coding region is interrupted by a type II intron of 850 nucleotides and a second intervening sequence of 357 nucleotides. Genomic sequence rearrangement within the first intron creates a nontranscribed partial copy of the gene. The intact ND 5 gene is transcribed in a complex pattern with mRNAs including the 5 S rRNA sequence. Excision of the two introns appears to proceed slowly in vivo since the steady state mitochondrial RNA contains significant proportions of unprocessed precursor molecules.

Isolation of major heat shock protein (hsp70) gene-related sequences from rat genome.

Rat genome was assayed for the presence of hsp70 gene-related sequences. Southern blots prepared from rat DNA digested with EcoRI or HindIII restriction endonucleases were hybridized with mouse, human and fruit fly hsp70 gene probes at increasing stringencies. At the stringency which allows sequences divergent up to about 30% to form stable complexes all three probes detected 25-30 restriction fragments. Increased stringency of the hybridization reduced the number of detectable bands to a few and among them the DNA fragments hybridizing specifically either with mouse or human hsp70 gene probes were detected. Most of the genomic fragments containing hsp70 gene-related sequences were subsequently isolated by screening the rat genomic library with mouse hsp70 gene probe. 168 positive clones were plaque purified and on the basis of the restriction and hybridization pattern we deduced that inserts represented 20 different genomic regions. Partial restriction maps of all isolated genomic fragments were constructed and regions containing hsp70 gene related as well as highly repetitive DNA sequences were localized. A putative sequence rearrangement in the proximity of the hsp70 gene-related sequence was detected in one of the isolated genomic segments.

A study of the structure of the gene for lecithin: cholesterol acyltransferase in four unrelated individuals with familial lecithin: cholesterol acyltransferase deficiency.

1. We have used polyclonal antibodies and a complementary DNA clone for human lecithin:cholesterol acyltransferase (LCAT) to study LCAT protein and the structure of the LCAT gene, respectively, in patients with familial LCAT deficiency from Norway, Ireland, Germany and Italy. 2. The patients had low levels of non-functional LCAT protein in their serum as measured by rocket immunoelectrophoresis; its mol. wt. of approximately 68,000 was identical with that of LCAT in normal plasma, as judged by immunoblotting. 3. Enzymatic digestion of DNA samples from the patients produced LCAT gene fragments which were indistinguishable from those found in normal individuals. 4. We conclude that LCAT deficiency in these patients is not caused by a large deletion or rearrangement of the LCAT gene sequences.

Transcription factors mediate rRNA synthesis during myogenesis.

This study was designed to identify factors which control rRNA transcription during terminal differentiation of rat L6 myoblasts. A cell-free system using 100,000 x g supernatants (S100 extracts) from rat L6 myoblasts and a genomic rRNA clone was established which accurately transcribes the rat rRNA gene. The myoblast S100 extract produced a high level of transcription with accurate initiation at the rRNA promoter. Myotube S100 extracts, on the other hand, displayed a reduced capacity to transcribe the rRNA gene. Thus constituents of the cell extract, rather than rearrangements of the DNA sequences, are implicated in regulating rRNA gene expression. The RNA polymerase content of the S100 extracts was analyzed using alpha-amanitin to determine the activity of each polymerase species and found to be comparable in both myoblast and myotube S100 extracts. This observation suggests that factors other than RNA polymerase I are involved in regulating rRNA transcription. Two of the factors associated with accurate rRNA gene expression in vitro, namely transcription factors B and D, occurred at comparable levels in both myoblast and myotube extracts suggesting that other components of the transcription complex are responsible for limiting rRNA transcription in myotubes. The cell-free system should facilitate identification and characterization of these factors which regulate expression of the rRNA gene.

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

Expression and biosynthetic variation of the epidermal growth factor receptor in human hepatocellular carcinoma-derived cell lines.

Expression of the epidermal growth factor (EGF) was analyzed in six human hepatocellular carcinoma-derived and one human hepatoblastoma-derived cell line, each of which retained the differentiated phenotype and functions of the parenchymal hepatocyte. The level of receptor expression of each hepatoma cell line was similar to that of the normal human fibroblast, approximately 10(5) molecules per cell. However, NPLC/PRF/5, a subline of the PLC/PRF/5 cell line obtained following reestablishment of a xenograft tumor in vitro, was found to express 4 x 10(6) high-affinity EGF receptor molecules per cell. Proliferation of the NPLC/PRF/5 cell line was inhibited in the presence of nanomolar quantities of ligand. Receptor overexpression was found to result from EGF receptor gene amplification without apparent rearrangement of the EGF receptor coding sequences. Although cell-specific variability in posttranslational processing of EGF receptor N-linked oligosaccharides in the hepatoma cell lines was found, no difference between the receptors in PLC/PRF/5 and NPLC/PRF/5 was observed and no aberrant receptor-related species were detected. EGF receptor gene amplification in the NPLC/PRF/5 cell line is probably a reflection of genome instability and selection of variants with augmented growth potential in limiting concentrations of EGF in vivo. When viewed in this light, EGF receptor overexpression could represent a manifestation of tumor progression in the EGF-responsive hepatocyte.

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants.

The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene. The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene.

Multiple sequence rearrangements accompanying the duplication of a tRNAPro gene in wheat mitochondrial DNA

In the course of isolating tRNA genes from wheat mtDNA, we have found the same tRNA(Pro) gene in two different Hind III restriction fragments, H-P1 (0.7 kbp) and H-P2 (1.7 kbp). Sequences immediately flanking these duplicate genes are closely related, although not identical; sequence comparisons suggest that multiple rearrangements have occurred in the vicinity of the H-P2 tRNA(Pro) gene, relative to the H-P1 version. The chimeric nature of H-P2 is emphasized by the presence of sequences that are also found upstream of the wheat mitochondrial 26S rRNA gene, as well as sequences derived from chloroplast DNA. Comparison of H-P2 with H-P1 plus upstream sequences provides some insight into possible molecular events that might have generated H-P2. In particular, such comparisons suggest a model in which the homologous sequences in H-P2 are seen to be derived from H-P1 plus upstream sequences as a result of an intragenomic, site-specific rearrangement event, followed by amplification of the product, its fixation in the mitochondrial genome, and subsequent sequence divergence (single base changes as well as insertions/deletions of up to 50 nucleotides). The results reported here implicate particular primary sequence motifs in certain of the rearrangements that characterize H-P2.

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.

Nature of recombination involved in excision and rearrangement of human repetitive DNA

An alphoid-like human repetitive DNA of the Sau3A family is present extrachromosomally and in the chromosomes. In the chromosomes, the DNA is located on chromosome 11 but related sequences are present in chromosome 17. We characterized the nature of the recombination involved in the excision of the extrachromosomal DNA from chromosome 11. The results show that the recombination occurs both between the homologous subunits and between the heterologous subunits with only a 70 to 80% sequence homology among them, suggesting that a DNA structure other than a sequence homology mediates the recombination process. The same type of recombination is responsible for the rearrangement of the related sequences in chromosome 17.

Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes.

In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA. One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC. Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro. Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint. These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs. Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.

EVOLUTION OF ANIMAL MITOCHONDRIAL DNA: RELEVANCE FOR POPULATION BIOLOGY AND SYSTEMATICS

In the past decade, mitochondrial DNA (mtDNA) analysis has become established as a powerful tool for evolutionary studies of animals. These studies, recently reviewed by Avise (8) and Wilson et al (149), have used mtDNA analyses to provide insights into population structure and gene flow, hybridization, biogeography, and phylogenetic relationships. Major advances have been made in our understanding of the molecular biology of animal mtDNA (reviewed in 7, 28). A powerful synergism exists between the two fields of research: Evolutionary studies provide comparative data on mtDNA organization and function, and molecular investigations can, and should, improve the level of sophistication of evolutionary studies that use mtDNA. Here we focus on molecular aspects of animal mtDNA that are especially relevant to its use in evolutionary studies. Thus, we consider the form and frequency of three types of change in mtDNA: base substitution, length variation, and sequence rearrangement. Attention is also given to the nature and possible consequences of interactions between nuclear and mitochondrial (mt) genes. In the concluding sections, we discuss the way in which the knowledge of molecular processes allows informed use of mtDNA variation in evolutionary studies. Only animal mtDNAs are considered. The molecular biology and evolution of chloroplast DNA and nonmetazoan mtDNAs have been reviewed elsewhere (41, 66, 111, 121, 123).

Mutation of a neutral amino acid in the transit peptide of rat mitochondrial malate dehydrogenase abolishes binding and import.

We have investigated the function of a leucine residue in the transit peptide of the rat mitochondrial malate dehydrogenase precursor using in vitro mutagenesis. Amino acid replacement of leucine 13 with glutamic acid and asparagine abolished import into mitochondria, while substitutions with proline, histidine, and arginine severely diminished uptake. In contrast, glutamine, tyrosine, valine, and alanine replacement resulted in normal levels of import, suggesting that there is a requirement for an uncharged residue at this position. Mutants involving rearrangements of the native sequence at positions 12-14 were imported as efficiently as the wild-type mitochondrial malate dehydrogenase, indicating that there was not an obligatory order of amino acid residues. However, deletion of leucine 13 resulted in diminished import. Binding studies with isolated mitochondria revealed that several position 13 mutants were deficient in binding to the mitochondrial surface, accounting for the reduced import of these proteins. This impairment could be distinguished from the effects due to decreased positive charge. We conclude that while translocation depends on the net positive charge, binding to the mitochondrial surface is mediated by uncharged residues within the transit peptides of mitochondrial precursor proteins.

Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila.

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3′ half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.