Amplification Mutants Research Articles

Gene amplification mutations originate prior to selective stress in Acinetobacter baylyi.

The controversial theory of adaptive amplification states gene amplification mutations are induced by selective environments where they are enriched due to the stress caused by growth restriction on unadapted cells. We tested this theory with three independent assays using an Acinetobacter baylyi model system that exclusively selects for cat gene amplification mutants. Our results demonstrate all cat gene amplification mutant colonies arise through a multistep process. While the late steps occur during selection exposure, these mutants derive from low-level amplification mutant cells that form before growth-inhibiting selection is imposed. During selection, these partial mutants undergo multiple secondary steps generating higher amplification over several days to multiple weeks to eventually form visible high-copy amplification colonies. Based on these findings, amplification in this Acinetobacter system can be explained by a natural selection process that does not require a stress response. These findings have fundamental implications to understanding the role of growth-limiting selective environments on cancer development. We suggest duplication mutations encompassing growth factor genes may serve as new genomic biomarkers to facilitate early cancer detection and treatment, before high-copy amplification is attained.

Analysis of IS 1236 -Mediated Gene Amplification Events in Acinetobacter baylyi ADP1

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.

Genome‐wide selection for increased copy number in Acinetobacter baylyi ADP1: locus and context‐dependent variation in gene amplification

Renewed interest in gene amplification stems from its importance in evolution and a variety of medical problems ranging from drug resistance to cancer. However, amplified DNA segments (amplicons) are not fully characterized in any organism. Here we report a novel Acinetobacter baylyi system for genome-wide studies. Amplification mutants that consume aromatic compounds were selected under conditions requiring high-level expression from three promoters in a linked set of chromosomal genes. Tools were developed to relocate these catabolic genes to any non-essential chromosomal position, and 49 amplification mutants from five genomic contexts were characterized. Amplicon size (18-271 kb) and copy number (2-105) indicated that 30% of mutants carried more than 1 Mb of amplified DNA. Amplification features depended on genomic position. For example, amplicons from one locus were similarly sized but displayed variable copy number, whereas those from another locus were differently sized but had comparable copy number. Additionally, the importance of sequence context was highlighted in one region where amplicons differed depending on the presence of a promoter mutation in the strain from which they were selected. DNA sequences at amplicon boundaries in 19 mutants reflected illegitimate recombination. Furthermore, steady-state duplication frequencies measured under non-selective conditions (10(-4) to 10(-5) ) confirmed that spontaneous gene duplication is a major source of genetic variation.

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

Gene Amplification Involves Site-specific Short Homology-independent Illegitimate Recombination in Acinetobacter sp. Strain ADP1

A system for studying gene amplification in the bacterium Acinetobacter sp. strain ADP1 was used to isolate 105 spontaneous mutants. The method selects for the elevated expression of neighboring transcriptional units in a parent strain lacking its normal transcriptional activators. Gene amplification can compensate for the activator loss by increasing the copy number of seven weakly expressed genes. Mutant colonies arose from the parent strain at a frequency of 10 −8 within three weeks. All but one of these mutants carried tandem head-to-tail repeats of a chromosomal segment (amplicon). These amplicons varied in size from approximately 12–290 kb and ranged in copy number from 3 to more than 30. Gene amplification involved a two-step process in which duplications formed independently of recA. Illegitimate recombination fused normally distant chromosomal regions to create novel DNA duplication junctions. These junctions were isolated from amplification mutants using an assay that exploits Acinetobacter natural transformability. Sequence analysis of 72 junctions revealed little identity in the recombining regions. Furthermore, multiple independently isolated mutants contained identical junctions. Six different junctions, each found in two to six mutants, revealed that some recombination events are site-specific. Several recurring junctions were studied using PCR. In each case, the identical duplication present in the mutant was estimated to have occurred in as many as one in a million cells in populations of strains never exposed to selective conditions. These duplications appeared to form spontaneously by a novel type of short homology-independent, site-specific process. However, in the absence of recA, mutant colonies were not selected from parent cells containing these duplications. Thus, the second gene amplification step most likely depends on homologous recombination to increase amplicon copy number. These studies support the theory that gene amplification is a driving force in the evolution of functionally related gene clusters.

Unstable retrovirus mutants with acquired transforming activity: rapid changes in the number of repeats of a specific junD polynucleotide segment.

We have previously reported that the non-transforming jun D (wild type) gene can acquire transforming activity through spontaneous mutations when it is replicated through avian replication-competent retrovirus vectors in chicken embryo fibroblasts. In two of these spontaneous mutants, T1 and T2, which were isolated from proviral DNA in the same transformed cell clone, a specific 48 bp polynucleotide segment of the jun D coding sequence was tandemly repeated three and five times, respectively. We report here that the number of direct repeats in these mutants rapidly changes (mostly decreases) in the context of either RSV-based replication-competent or MLV-based replication-defective retroviruses, most likely during the process of reverse transcription, while these mutations are stable in the cellular chromosome. We also show that the growth conditions of the infected culture modulate the proportions of polymorphic proviral populations in the infected culture. We finally discuss the possible molecular mechanisms that generate genetic diversity in these amplification mutants.

Mutational analysis of the sequence-specific recombination box for amplification of gene 17 of bacteriophage T4

Bacteriophage T4 gene 17 amplification mutants Hp17 that carry two to six tandem repeats of the genes 17-18 region were isolated by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli. These mutants arise from an initial sequence-specific recombination between two GCTCA sequences in a 24 bp imperfect homology box in genes 16 and 19. The initial recombination occurred in the wild-type phage T4 population, as shown by polymerase chain reaction, at a frequency of about 10(-6), which is consistent with the frequency of mutant isolation. T4 phage with mutations of the 3rd, 6th, 9th, 12th, or 15th positions in the 24 bp box of gene 16 either failed to produce gene amplification mutant Hp17 or produced gene amplification mutants from an initial recombination at other regions. Among the mutants that failed to produce gene amplification mutants, the initial recombination generally occurred at lower frequencies at either the GCTCA sequence or other sequences. Since the gene amplification mutations are eliminated or shifted to different sequences by base changes that increase as well as decrease homology, the predominant recombination event between the gene 16 and 19 recombination boxes appears to be sequence-dependent rather than homology-dependent.

Bacteriophage T4 gene 17 amplification mutants: Evidence for initiation by the T4 terminase subunit gp16

Bacteriophage T4 genes 16 and 19 containing the 24 bp homology regions that recombine to form Hp17 mutants were cloned into plasmids. When the two homology sequences were cloned either together into one or separately into two compatible plasmids, a polymerase chain reaction assay showed that recombination occurred in vivo. The recombinant sequence was identical with that found in T4 phage Hp17 mutants, and was produced in recombination-deficient Escherichia coli. Mutational analysis revealed a requirement for functional gene 16 but not gene 17 to recombine the sequences. Moreover, gp16, the terminase small subunit, was required, since an amber gene 16 produced the recombinant sequence only when suppressed. Mutations in the gene 16 recombination sequence (3GA and 15TG) that eliminated Hp17 formation in T4 phage increased the synthesis of the large terminase subunit, gp17 in T4 infections, suggesting gp16 interaction with this site. gp16 binding to gene 16 and gene 19 pac-like sites may synapse the homologous sequences to lead to Hp17 mutant formation, and this suggests a synapsis mechanism for control of T4 DNA maturation and concatemer processing in packaging.

Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants

Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17–18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TagI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt − heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4Hp17 amplification mutants.

Generation of metastatic variants in populations of mutator and amplificator mutants.

Genetic instability has been hypothesized by P. C. Nowell and other investigators to be an important aspect of tumor progression that leads to the generation of metastatic variants. In this study we examined the rate of generation of metastatic variants in mutant cell lines having increased rates of spontaneous mutation and gene amplification. Parallel clonal populations of the spontaneous mutation rate mutant thy-49 and the gene amplification mutants YMP1 and YMP7 and their respective wild types were generated and grown to a critical population size. The number of metastatic variants in each clonal population was then determined following iv injection into nude mice. Lung tumors were scored 3-4 weeks after injection of cells, and the mean number per clonal population was determined. Analysis of the means with the Luria-Delbruck fluctuation test showed no significant differences in the rate of generation of metastatic variants produced in the genetically unstable lines compared to their normal counterparts. This study suggests that increased spontaneous mutation and gene amplification rates in mammalian cells are not sufficient on their own to increase the rate of generation of metastatic variants.

Trans-acting amplification mutants and other eggshell mutants of the third chromosome in Drosophila melanogaster.

We report on the characterization of five third chromosome mutations with strong effects on the formation of the eggshell or chorion. Three mutations, defining two loci, result in substantially reduced follicle cell-specific amplification of the major chorion structural genes and, hence, in underproduction of the corresponding mRNAs and proteins. The other two mutations, though displaying structural chorion abnormalities, appear to have no significant effect on amplification and to express normally the major chorion structural genes. The possible nature of these mutations is discussed.

Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

Selective overproduction of adenosine deaminase in cultured mouse cells.

The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.