On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1).

A wide spectrum of mutations, ranging from point mutations to large deletions, have been described in the retinoblastoma gene (RB1). Mutations have been found throughout the gene; however, these genetic alterations do not appear to be homogeneously distributed. In particular, a significant proportion of disease-causing mutations results in the premature termination of protein synthesis, and the majority of these mutations occur as C-->T transitions at CpG dinucleotides (CpGs). Such recurrent CpG mutations, including those found in RB1, are likely the result of the deamination of 5-methylcytosine within these CpGs. In the present study, we used the sodiumbisulfite conversion method to detect cytosine methylation in representative exons of RB1. We analyzed DNA from a variety of tissues and specifically targeted CGA codons in RB1, where recurrent premature termination mutations have been reported. We found that DNA methylation within RB1 exons 8, 14, 25, and 27 appeared to be restricted to CpGs, including six CGA codons. Other codons containing methylated cytosines have not been reported to be mutated. Therefore, disease-causing mutations at CpGs in RB1 appear to be determined by several factors, including the constitutive presence of DNA methylation at cytosines within CpGs, the specific codon within which the methylated cytosine is located, and the particular region of the gene within which that codon resides.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Point mutation of the p53 gene resulting in splicing inhibition in small cell lung carcinoma

The p53 gene is functionally inactivated mostly by point mutations resulting in amino acid substitutions in a wide variety of human cancers. We found a novel mutation of the p53 gene in a small cell lung carcinoma cell line, Lu-143. One of the allelic p53 genes was lost accompanied by loss of heterozygosity for chromosome 17. In the remaining allelic p53 gene, there was a single-base substitution of G to T at position 1 within the splice donor site of intron 7, and the mutated intron was not spliced out during the mRNA maturation process. As a result of this mutation, larger sized p53 mRNA was expressed and no p53 specific protein was detected in this cell line. These results suggest that mutations causing splicing abnormalities are one of the molecular mechanisms for the p53 gene inactivation in human cancer.     

Needle-in-a-haystack detection and identification of base substitution mutations in human tissues

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10⁷ wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10⁶ wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10⁻⁵ and 10⁻⁶. These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10⁻² to 10⁻⁷) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.     

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.     

Components required for cytokinesis are important for bud site selection in yeast

Polarized cell division is a fundamental process that occurs in a variety of organisms; it is responsible for the proper positioning of daughter cells and the correct segregation of cytoplasmic components. The SPA2 gene of yeast encodes a nonessential protein that localizes to sites of cell growth and to the site of cytokinesis. spa2 mutants exhibit slightly altered budding patterns. In this report, a genetic screen was used to isolate a novel ochre allele of CDC10, cdc10-10; strains containing this mutation require the SPA2 gene for growth. CDC10 encodes a conserved potential GTP-binding protein that previously has been shown to localize to the bud neck and to be important for cytokinesis. The genetic interaction of cdc10-10 and spa2 suggests a role for SPA2 in cytokinesis. Most importantly, strains that contain a cdc10-10 mutation and those containing mutations affecting other putative neck filament proteins do not form buds at their normal proximal location. The finding that a component involved in cytokinesis is also important in bud site selection provides strong evidence for the cytokinesis tag model; i.e., critical components at the site of cytokinesis are involved in determining the next site of polarized growth and division.     

Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites

Two related carcinogen adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) or N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into the lacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA. The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct. These site-specifically modified templates were transfected into competent wild-type Escherichia coli cells (JM103) and a uvrA strain (SMH12). The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective beta-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region. DNA sequencing was used to characterize 179 of the mutants obtained. We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct. A specific frameshift (+1G) was also observed at a displaced site. All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide. Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts. The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression.     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Nucleotide sequence analysis of novel junctions near an unstable integrated plasmid in human cells

Characterization of human cell clones containing a promoterless selectable gene (neo), integrated at various locations in the genome, demonstrated that one of the integration sites had a high rate of spontaneous tandem duplications. Other investigators have suggested that specific sequences, such as short repeats, found near an integration site, could be responsible for this kind of instability. To learn more about this process, we sequenced the DNA at the recombination site in two independently derived subclones, and compared these sequences with those found in the parental cell DNA. The results demonstrate that specific sequences are not required at the recombination site. In one G418-resistant subclone, recombination occurred between an Alu retroposon in the cellular DNA and integrated pBR322 sequences sharing 3 bp of similarity at the recombination site. In the other subclone, recombination occurred between single-copy cellular DNA and integrated simian virus 40 sequences sharing a single bp of similarity at the recombination site. This heterogeneity at the recombination site indicates a general enhancement of the rate of recombination within the entire region, with little if any sequence specificity or similarity required.     

Effect of upstream RNA processing on selection of mu S versus mu M poly(A) sites.

All of the regulatory factors responsible for augmenting microseconds mRNA levels preceding the dramatic increase in secretory IgM production upon B cell activation has not been totally elucidated. Whereas previous experiments have centered on the region of the gene specifying the choice between splicing to mu M exons versus selection of the mu S poly(A) site, we have found that upstream sequences within the Cmu gene, specifically the Cmu 4 acceptor splice site together with intronic sequences between the Cmu 3++ and Cmu 4 exons, play an important role in dictating the precision or the extent of splicing to the mu M exons even under conditions in which functional polyadenylation factors should be in excess. Therefore, splicing of upstream exons can affect remotely located downstream exons. These findings suggest that regulation of differential mu S/mu M mRNA expression may involve general processing enzymes that recognize specific cis -regulatory sequences residing within the body of the mu gene and account for the unique ability of activated B cells to secrete copious amounts of IgM.     

Molecular characterization of seven beta-thalassemia mutations in Asian Indians

To characterize systematically the mutations which produce beta-thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta-globin gene cluster of 44 beta-thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta-gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5' to the beta-globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5' to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5' and 3' to it, suggesting that it lies within a region of relative sequence randomization.     

Four Distinct Regulatory Regions of the Cut Locus and Their Effect on Cell Type Specification in Drosophila

The cut gene in Drosophila is necessary in at least one cell type, the external sensory organs, for proper cell type specification and morphogenesis. It is also expressed in a variety of other tissues, where its function is less well characterized. Previous work has demonstrated that mutations affecting all the tissues map in the transcribed and translated portion of the gene, while mutations that are tissue specific in their effects map in the 140 kb upstream of the most 5' exon known. Within that 140 kb, the mutations fall into four subregions, two of which contain mutations affecting unique sets of tissues and the other two of which contain mutations that affect a third set. Our examination of the defects of mutants, their complementation behavior, and their effect on the distribution of the cut protein in embryos, alters the picture in three important ways. First, some mutations convert the cells of the Malpighian tubules into what appear to be gut cells, suggesting that cut is necessary for cell type specification and morphogenesis in a variety of tissues. Second, mutations in each of the four subregions in the 140 kb of upstream DNA cause a different set of phenotypes, suggesting that the regulatory region contains at least four separate units with different tissue specific functions. And third, mutations have now been identified that map in the transcribed and translated portion of the gene but that have tissue specific effects.     

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.     

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.     

Mutational analysis of the proteolytic cleavage site of glycoprotein B (gB) of Marek''s disease virus

The Marek's disease virus (MDV) glycoprotein B (gB) precursor, gp100, is proteolytically cleaved into two disulfide-linked subunits, gp60 and gp49. In the gB homologs of most other herpesviruses, a tetrapeptide, Arg-Xaa-Arg-Arg, is immediately upstream from the predicted cleavage site. We have investigated the specificity of the proteolytic cleavage in gplOO by introducing mutations within its predicted cleavage site (Arg-Leu-Arg-Arg) and expressed these mutants in recombinant fowlpox virus (FPV). The results show that all three Arg residues at the predicted cleavage site play an important role in the specific proteolytic cleavage of gp100. Furthermore, we demonstrated that the cleavage of gplOO is not necessary for transport of gB to the cell surface.     

Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells

We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes.