PM2 bacteriophage plaque morphology mutants exhibit differences in DNA length and restriction endonuclease patterns

A large plaque (LP) and a small plaque (SP) variant of PM2 bacteriophage were isolated from a mixture of the two plaque variants and were grown separately in the appropriate host bacterium,Alteromonas espejiana. They have remained pure for approximately one year from the original isolation. Restriction endonuclease analyses revealed differences in theHaeIII restriction profile between the two variants.HaeIII fragment 1 of the SP DNA was found to be smaller than the corresponding fragment from the LP variant DNA, whereas fragment 7 from the SP DNA was slightly larger than the same fragment from LP DNA. Electron microscopy of heteroduplexes formed between the DNAs from the two variants revealed that the deletion in fragment 1 mapped very close to the junction betweenHaeIII fragments 1 and 13 on the physical map of PM2 DNA. The difference in DNA length between the two variants results from addition or deletion mutations.Deceased.     

A 4-base-pair deletion polymorphism in man

Partial nucleotide sequencing of C11p11, a probe mapping close to the gene determining familial adenomatous polyposis (FAP) on human chromosome 5, in 4 unrelated persons has revealed a 4-base-pair deletion variant designated DELI at an arbitrary DNA locus D5S71. For screening the deletion variants that may frequently occur in the non-coding DNA sequences, we set up a non-invasive procedure which involves DNA amplification by PCR, simple polyacrylamide gel electrophoresis and direct visualisation of alleles under long wave ultraviolet light by ethidium bromide staining.     

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.     

Construction of a viable SV40 variant containing two functional origins of DNA replication.

Viable variants of simian virus 40 (SV40) have been constructed which contain two functional origins of DNA replication (Or). The variants were made by introducing, at 0.175 on the SV40 map, a segment of DNA containing the viral Or. Two types of experiments demonstrate that the second Or is functional. First, the distribution of radioactivity in pulse-labeled SV40 (I) DNA is dramatically altered in the variants when compared with the parental virus. Second, electron microscopic examination of viral replicative intermediates indicates that while there is one initiation site for DNA synthesis in the parental genome, there are two sites in the variant. It was possible to introduce a deletion which inactivated the original Or at 0.67 map units in this variant. The resulting mutant could be propagated, and its DNA replication originated at the site of the newly inserted Or.     

Variant forms of DNA polymerase beta in primary lung carcinomas.

DNA polymerase beta (pol beta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. A truncated form of the pol beta protein is expressed in colon and breast cancers. However, the role of the pol beta gene in lung cancer is not known. Thus, we investigated a possible occurrence of pol beta variants in primary lung tumors. The entire cDNA of pol beta obtained by RT-PCR amplification was analyzed for nucleotide sequencing in lung tumor and matched normal lung tissue of the same patient. Three types of variants were detected in squamous, non-small, or large cell carcinomas. The most common variant was a deletion of 87 bp from pol beta cDNA at a site corresponding to exon 11. In addition, a variant exhibiting deletions of 87 and 140 bp together with an insertion of 105 bp was identified in three lung tumors. This is the first report of the occurrence of pol beta variants, possibly splicing variants, in lung cancer. A truncated pol beta protein resulting from variant forms of the gene may impact the function of the enzyme and increase susceptibility to carcinogenesis.     

Genetic diversity of moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of kappa-Casein and exon 2 of MhcALal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075-0.15 Myr ago.     

Insertion–Deletion Polymorphism of Mitochondrial DNA Region V in Kazakh Populations from Different Regions of Kazakhstan

Insertion–deletion polymorphism of the mitochondrial DNA (mtDNA) region V was examined in three Kazakh populations inhabiting different regions of Kazakhstan. The 9-bp deletion was revealed in all three populations examined. In Altai population the 4-bp insertion was also found. The presence of these polymorphic variants was confirmed by DNA sequencing.     

Characterization of form variants of Xenorhabdus luminescens.

From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed.     

Characterization of form variants of Xenorhabdus luminescens.

From Xenorhabdus luminescens XE-87.3 four variants were isolated. One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red). A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye. Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white). Both were less luminescent, did not take up dye, and had small cell and colony sizes. These two variants were very unstable and shifted to the primary form after 3 to 5 days. It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant. The white variant was also found in several other X. luminescens strains. DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent. Protein patterns revealed a few differences among the four variants. None of the variants could be considered the secondary form. The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white. The mechanism and function of this variability are discussed.     

Genetic Diversity of Moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian, and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species origin or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of -Casein and exon 2 of MhcAlal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075–0.15 Myr ago.     

Development of molecular approaches to estimating germinal mutation rates. I. Detection of insertion/deletion/rearrangement variants in the human genome

DNA from 130 individuals was studied with up to 18 (primarily cDNA) probes for the frequency of variants in this initial experiment to determine the feasibility of this approach to screening for germinal gene mutations. This approach, a modification of the usual restriction enzyme mapping strategy, focuses on the detection of insertion/deletion/rearrangement (I/D/R) variants, because the DNA is digested with only two restriction enzymes before transfer to membranes and hybridization with an extensive series of unrelated probes. Some 4000 noncontiguous, independent DNA fragments ("loci"), functional loci, pseudogenes or anonymous fragments, (a total of approximately 77,400 kb) were screened. 19 different classes and 31 copies of presumably I/D/R variants were detected while 4 different classes and 24 individuals exhibiting base substitution variants were observed. 18 of the 19 I/D/R classes were rare variants, that is, each were observed at a frequency, within this population, of less than 0.01; 3 of the base substitution classes existed at polymorphic frequencies and only 1 was a rare variant. 10 of the I/D/R classes, occurring in a total of 18 individuals, were detected with probes which are not known to be associated with repetitive elements. This is a variant frequency for I/D/R variants without known repetitive elements of 0.15 classes and 0.23 copies for each 1000 kb screened; this would extrapolate to 1600 such variant sites in the genome of each individual. Within the context of a mutation screening program, the rare variants, either with or without repetitive elements, would have a higher probability of being de novo mutations than would polymorphic variants; this former group would be the focus of family studies to test for the heritability of the allele (fragment pattern). Sufficient DNA probes are available to screen a significant portion of the human genome for genetic variation and de novo mutations of this type.     

Insertion-deletion polymorphism of mitochondrial DNA region V in Kazakh populations from different regions of Kazakhstan

Insertion-deletion polymorphism of the mitochondrial DNA (mtDNA) region V was examined in three Kazakh populations inhabiting different regions of Kazakhstan. The 9-bp deletion was revealed in all three populations examined. In Altai population the 4-bp insertion was also found. The presence of these polymorphic variants was confirmed by DNA sequencing.     

Arrangement of the genome of the human papovavirus RF virus.

DNA from plaque-purified RF virus, a variant of BK virus, was found to contain two species of molecules. Hybridization of each DNA species to the fragments of BK virus DNA revealed that one species had a deletion corresponding to at least 50% of the late region and the other had a deletion corresponding to at least 40% of the early region of BK virus DNA. Analysis by cleavage of each RF virus DNA species with restriction endonucleases EcoRI, HindIII, AvaII, and PvuII, when compared with BK virus DNA, revealed that the size and number of fragments were different. These results suggest the loss of some restriction sites and the appearance of new sites, probably as a result of base changes in each RF virus DNA species. Furthermore, analysis of the restriction map of each DNA molecule revealed in insertion(s) in both DNA species.     

Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0.1-100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rare-cutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II variants have reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type II variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability.

Plasmid R68.45 has been useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosome-mobilizing ability (Cma) may be lost without the loss of other plasmid markers. However, Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). We isolated variants of R68.45 from four bacterial species of three genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma, and Km resistance. In Erwinia carotovora subsp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. Furthermore, the types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. Therefore, we suggest that the DNA duplication is the source of much of the instability observed in R68.45. However, data are presented for E. carotovora subsp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species.     

Plasmodium falciparum: comparison of the genomic organization of the knob protein gene in knobby and knobless variants

The genomic organization of the knob protein (KP) gene of knobby (K+) and knobless (K-) variants of the Thai isolate NT 108 and the Gambian isolate FCR-3 are compared. The restriction enzyme maps and the chromosomal location of the KP gene of K+ variants of both isolates are apparently identical. A comparison of the susceptibility of the KP gene to deletions and the extent of deletion of chromosomal DNA in K- variants of each geographical isolate suggests isolate-specificity of the stability of the deleted DNA sequences. With the exception of a mutant K- population of FCR-3, which could not be distinguished from K+ FCR-3, in all other K- variants of both geographical isolates the deletion of the KP gene was accompanied by the loss of several hundred base pairs of DNA from chromosome 2. The deletion resulted in the localization of the mutant KP gene in proximity to telomeric DNA sequences.