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PubMed1990

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.

Base Sequence↗
PubMed1990

Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein.

The Tat transactivator protein of human immunodeficiency virus type 1 contains a highly conserved cysteine-rich region, containing seven cysteines from residues 22 through 37. To investigate the importance of noncysteine residues in this region of the Tat protein, we have carried out a mutational analysis, in most cases substituting a single alanine for the wild-type noncysteine residue. Alanine substitution of residue 23, 24, 46, or 47 had no effect on Tat activity in plasmid transfection assays. In contrast, alanine substitutions of all eight noncysteines analyzed, from residues 26 through 41, significantly reduced the activity of the Tat protein, in some cases as drastically as mutations in cysteine residues. The results demonstrate that the precise sequence of the cysteine-rich region is crucial for a fully functional Tat protein.

Amino Acid Sequence↗
PubMed1992

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.

Animals↗
PubMed1991

Analysis of Salmonella typhimurium hisD3052 revertants: the use of oligodeoxyribonucleotide colony hybridization, PCR, and direct sequencing in mutational analysis.

A rapid method for determining the DNA sequences of Salmonella typhimurium hisD3052 revertants is presented. DNA colony hybridization was used to analyze revertants previously studied by Isono and Yourno [Proc Natl Acad Sci USA 71:1612-1617, 1974]. Synthetic oligodeoxyribonucleotide probes (18-mers) were able to distinguish sequences that differed by a single base pair. Mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (PCR) and directly sequenced. The combined use of DNA-colony hybridization and direct sequencing offers a precise and rapid means for the molecular characterization of hisD3052 revertants.

Base Sequence↗
PubMed1986

Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.

HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of the structure and function of GTP-binding proteins.

Structural, biochemical and molecular genetic studies of EF-Tu, p21ras and alpha s have begun to reveal the inner workings of the molecular machine used by these and other GTP-binding proteins. Further understanding of this molecular machine will ultimately come from crystal structures of the G protein alpha chains as well as from crystal structures of the GTP-bound forms of p21ras and EF-Tu. Mutational analysis will continue to add meaning to the static pictures provided by these crystal structures. Aside from their intrinsic biological interest, other reasons motivate our exploration of the GTP-dependent molecular machine used by GTP-binding proteins. Mutations or bacterial toxins cause disease by inhibiting the GTPase function of p21ras and alpha s. Other G protein alpha chains carry signals that regulate important cell functions, including proliferation. Malfunctions of these other G proteins are highly likely to cause disease. Applying our knowledge of p21ras and alpha s to these additional proteins may turn out to have significant practical consequences.

Animals↗
PubMed1992

Point mutational analysis of the hamster dihydrofolate reductase minimum promoter.

We have shown previously that 48 base pairs (bp) of 5'-flanking sequence are necessary for correct initiation at the major transcriptional start site of the Chinese hamster dihydrofolate reductase (dhfr) gene (Ciudad et al., 1988). As an upstream element, this sequence alone confers 25% of maximum promoter activity. The 5' half of this sequence is particularly well conserved among mammalian species; it contains one Sp1 binding site (GC box) and one CAA element. In the present work, we have analyzed the role of this region by extensive point mutational analysis. Twenty-three dhfr minigene constructs containing 1- or 2-base substitutions in this region of the promoter were tested by measuring their ability to transfect DHFR-deficient Chinese hamster ovary cells to a DHFR+ growth phenotype. Eight mutants, all in or near the GC box, exhibited reduced transfection efficiency. Promoter disfunction in these mutants was confirmed by RNase protection analysis of stable transfectants. Gel retardation experiments showed that mutants affected in the consensus sequence for Sp1 binding were deficient in binding a protein found in nuclear extracts of Chinese hamster ovary cells. Purified human transcription factor Sp1 was also unable to bind a promoter sequence bearing one of these single base substitutions, suggesting that Sp1 itself is involved in dhfr transcription in vivo. We conclude that most single base mutations in the GC box severely cripple or eliminate promoter function by inhibiting binding of transcription factors to this regulatory sequence and that Sp1 is likely to be involved in dhfr transcription in vivo. We also found that the well conserved CAA element is not absolutely necessary for transcription.

Animals↗
PubMed1990

Mutational analysis of the epidermal growth factor-receptor kinase.

The biological responses of epidermal growth factor (EGF) are mediated by a surface receptor denoted as the EGF receptor. The EGF receptor possesses intrinsic protein tyrosine kinase activity which is essential for signal transduction. Recent evidence shows that EGF receptor phosphorylates several substances including: phospholipase C-gamma and the GTPase-activating protein (GAP). Moreover, these proteins become associated with the activated receptor in an immunocomplex. Autophosphorylation of the EGF receptor appears to be required for the association with phospholipase C-gamma. Mutational analysis indicates that the intrinsic autophosphorylation sites compete with exogenous substrates for the substrate-binding site in the kinase domain. The ligand-binding site for EGF was analysed using a chimeric receptor approach. Subdomains of the extracellular ligand-binding region of the chicken EGF receptor, which binds EGF with low affinity, were replaced by corresponding regions of the human EGF receptor, which binds EGF with high affinity. On the basis of this analysis, it is concluded that subdomain III of the extracellular domain of the EGF receptor is a major ligand-binding domain. Together, domain I and domain III are able to reconstitute nearly all interactions which bring about high-affinity binding. Growth factor receptors with protein tyrosine kinase (PTK) activity could be envisioned as membrane-associated allosteric enzymes. Unlike water-soluble allosteric enzymes, the configuration of the growth factor receptors dictates that the ligand-binding domain and PTK activity of the receptor molecules are separated by the plasma membrane. Therefore, ligand-induced signal must cross the membrane barrier to activate the PTK function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Mutational analysis of att554, the target of the site-specific transposon Tn554.

Tn554 is a high-frequency, site-specific transposable element of Staphylococcus aureus which has integrative properties resembling those of temperate bacteriophages. Tn554 inserts at a unique chromosomal location, designated att554. att554 contains a core hexanucleotide sequence, 5'-GATGTA-3' (nucleotides numbered -3 to +3). Most of the time (greater than 99%) insertion occurs immediately 3' to this sequence; the resulting orientation of Tn554 to att554 is designated as the (+) orientation. Infrequent insertions immediately 5' to the core sequence result in the opposite, or (-) orientation. Mutational analysis of a cloned att554 site indicates that deletions extending from the left and ending at -15 or from the right ending between +8 and +12 reduced attachment site efficiency. Plasmids with deletions extending closer to the insertion site, although still retaining the core sequence from -3 to +3, were totally inactive. Tn554 insertions into partially active att554 sites retained normal site- and orientation-specificity with respect to att554, but they frequently contained abnormal sequences at the junction of att554 and the 3' end of Tn554. These data indicate that att554 contains a short nucleotide sequence essential for transposition and flanking sequences that greatly increase the frequency of recombination.

Base Sequence↗
PubMed1992

Mutational analysis of the tobacco etch potyviral 35-kDa proteinase: identification of essential residues and requirements for autoproteolysis.

The tobacco etch potyvirus (TEV) polyprotein is processed by three virus-encoded proteinases, termed Nla, HC-Pro, and the 35-kDa proteinase. The 35-kDa proteinase is derived from the amino-terminal region of the polyprotein. Analysis of polyproteins containing beta-glucuronidase fused to the expected carboxy terminus of the 35-kDa proteinase confirmed the previously identified Tyr304-Ser305 dipeptide as the cleavage site between the 35-kDa proteinase and HC-Pro. The 35-kDa proteinase of TEV was unable to catalyze proteolysis when synthetic substrate polyproteins were supplied in a bimolecular or trans reaction, suggesting that processing occurs by an autolytic mechanism. The results of a mutational analysis within the 35-kDa proteolytic domain indicated that His214, Asp223, Ser256, and Asp288 were required for optimal autoproteolytic activity. Replacement of Ser256 with either Thr or Cys resulted in low but detectable proteinase activity, as did substitution of Asp223 and Asp288 with Glu. These results are consistent with the hypothesis that the 35-kDa proteinase resembles cellular serine-type proteinases, with Ser256 functioning as the nucleophilic residue within the active site. Cleavage mediated by the 35-kDa proteinase has been shown previously to occur after polyprotein synthesis in wheat germ extracts and transgenic plants, but not in rabbit reticulocyte lysate. We were able to demonstrate that processing in vitro may require a heat-labile factor present in wheat germ extracts.

Amino Acid Sequence↗
PubMed1991

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis.

45 mutations (insertion, deletion and base substitution) of the G4 Goric were tested for their functional activity in M13 and R199 in vivo. The critical mutants were also assayed for their ability to synthesize pRNA in vitro using SSB and primase. The results demonstrate that the secondary structure and spacing of stem-loops I and III are essential for Goric activity and that the 5'-CTG-3' sequence flanking stem-loop I is essential for initiation of pRNA synthesis.

Bacteriophages↗
PubMed2014

Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow.

BACKGROUND: Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. METHODS: We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. RESULTS: Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. CONCLUSIONS: Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

Bone Marrow↗
PubMed1990

Mutational analysis of human NRAS genes in malignant melanoma: rapid methods for oligonucleotide hybridization and manual and automated direct sequencing of products generated by the polymerase chain reaction.

Three methods to detect single base mutations in codon 61 of the human NRAS gene from human melanoma DNA are described and compared: oligonucleotide hybridization analysis and direct manual and automated sequence analysis. Point mutations are detected by oligonucleotide hybridization and direct manual and direct automated sequence analysis of in vitro amplified genomic DNA. Heterozygosity for mutant alleles is reliably detected by oligonucleotide hybridization and by direct manual, but not by direct automated, sequence analysis. Generating single-stranded DNA via "asymmetric polymerase chain reaction (PCR)" and utilizing alpha 35S-dATP as radiolabel for manual sequencing and fluorescent-dye labeled primers for automated sequencing (Applied Biosystems, Inc.), we can obtain sequence information from either strand. The use of several of these methodologies to detect single base changes in the human NRAS gene is illustrated. In addition, the use of these and other related techniques to define the involvement of RAS oncogenes in human melanomas more precisely is reviewed.

Automation↗
PubMed1992

Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange.

We have developed a system for testing mutations by plasmid exchange in the fission yeast Schizosaccharomyces pombe. This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S. pombe. Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA. Substitution of the U4 sequence in stem 1 of the U4/U6 interaction domain allows accumulation of the mutant U4, but does not support viability. Two sequences with homology to the Sm binding site are found in the 3' region of S. pombe U4; substitution of the 3' sequence of the two does not interfere with accumulation or function of U4, indicating that the 5' sequence is the functional Sm-binding site.

Base Sequence↗