Temperature-sensitive Mutation In Gene Research Articles

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans.

Genome-Wide Synthetic Lethal Screens Identify an Interaction Between the Nuclear Envelope Protein, Apq12p, and the Kinetochore in Saccharomyces cerevisiae

The maintenance of genome stability is a fundamental requirement for normal cell cycle progression. The budding yeast Saccharomyces cerevisiae is an excellent model to study chromosome maintenance due to its well-defined centromere and kinetochore, the region of the chromosome and associated protein complex, respectively, that link chromosomes to microtubules. To identify genes that are linked to chromosome stability, we performed genome-wide synthetic lethal screens using a series of novel temperature-sensitive mutations in genes encoding a central and outer kinetochore protein. By performing the screens using different mutant alleles of each gene, we aimed to identify genetic interactions that revealed diverse pathways affecting chromosome stability. Our study, which is the first example of genome-wide synthetic lethal screening with multiple alleles of a single gene, demonstrates that functionally distinct mutants uncover different cellular processes required for chromosome maintenance. Two of our screens identified APQ12, which encodes a nuclear envelope protein that is required for proper nucleocytoplasmic transport of mRNA. We find that apq12 mutants are delayed in anaphase, rereplicate their DNA, and rebud prior to completion of cytokinesis, suggesting a defect in controlling mitotic progression. Our analysis reveals a novel relationship between nucleocytoplasmic transport and chromosome stability.

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA+, related to the cellular DNA damage response.

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.

Bacteriophage P2: Genes Involved in Baseplate Assembly

The sequences of two previously defined tail genes, V and J, of the temperate bacteriophage P2, and those of two new essential tail genes, W and I, were determined. Their order is the late gene promoter, VWJI , followed by the tail fiber genes H and G, and a transcription terminator. The V gene product is the small spike at the tip of the tail, and the J gene product lies at the edge of the baseplate. The W gene product may be homologous to the product of gene 25 of T4 phage, which is part of the T4 baseplate. A temperature-sensitive mutation in gene V affects satellite phage P4 production more than it affects the production of P2 helper phage. P4 mutations that partially compensate for this defect of gene V lie in the P4 capsid size determination gene, sid .

Glucose-induced activation of plasma membrane H +-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis

Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H +-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H +-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H +-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H +-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H +-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H +-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H +-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H +-ATPase have a common initiation point.

Regulation of DNA Replication during the Yeast Cell Cycle

One central event that must occur exactly once per cell division cycle is the replication of the genomic DNA. One defining observation of the eukaryotic cell cycle is that the nuclear DNA replicates during a short period (the S phase) and that during that period each part of every chromosome is entirely replicated. Although there are many origins of DNA replication per chromosome, and it is known that not all of them are required to act in every cycle, nevertheless reinitiation in the same cycle at any one of them is never observed. For these reasons, it has long been assumed that the initiation of DNA replication is under very stringent control. When Hartwell et al. (1970) began systematic isolation of cell-division-cycle (cdc) mutants more than 20 years ago, it was expected that mutants specifically defective in DNA replication, especially those involved in initiation of DNA replication, would be prominent among the mutants that exhibit cdc phenotypes. Growing yeast cells show a morphology characteristic for the position they have reached in the cell cycle: The bud emerges just about the time that S phase begins and has reached its full size at the time that mitosis begins. Thus conditional-lethal mutants defective in DNA elongation were expected to arrest, under nonpermissive conditions, with a large bud. Indeed, temperature-sensitive mutations in genes specifying DNA polymerases (cdc2,

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30–40% of wild-type activity at 50°C compared to 32°C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism ( rnb-500 and ams-l) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.

Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1

The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.

Retroregulation of the synthesis of ribosomal proteins L14 and L24 by feedback repressor S8 in Escherichia coli.

Previous studies on regulation of the spc operon containing genes for ribosomal proteins have shown that S8, encoded by the fifth gene of the operon in Escherichia coli, is a translational repressor and regulates the synthesis of the third gene product (L5) and distal gene products by acting at a site near the L5 mRNA translation initiation site. We have now shown that S8 also regulates the synthesis of the first and second gene products (L14 and L24) of the operon by acting at the same mRNA target site--that is, the site located distal to sites coding for L14 and L24--and that mRNA degradation is involved in this retroregulation. It was shown that single base substitutions in the target site, which abolish repression of the synthesis of L5 and L5-distal gene products by S8, also cause derepression of L14-L24 synthesis. Inhibition of L14-L24 synthesis by S8 was also shown by overproducing S8 in trans from a plasmid carrying the S8 gene under lac promoter/operator control. A strain carrying temperature-sensitive mutations in genes for polynucleotide phosphorylase and RNase II was found upon shift to nonpermissive temperature to show higher differential synthesis rates of L14-L24 (and L5) relative to those of several L5-distal spc operon gene products. We suggest that repression of distal ribosomal protein synthesis by S8 triggers nucleolytic cleavage of spc operon mRNA, followed by mRNA degradation by these 3'- to 5'- exonucleases, which is then responsible for inhibition of L14-L24 synthesis.

Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations.

The "start" cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively). Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic "petite" mutants and catabolite repression resistant mutants.

Head morphogenesis of bacteriophage T4 I. Isolation and characterization of gene 40 mutants

Bacteriophage T4 gene 40 was classified originally as an essential gene for capsid formation because temperature-sensitive mutations in gene 40 result in polyhead production in the infected cells. Amber, ochre, and opal mutants in this gene have now been isolated and characterized and are found to grow with low efficiency on suppressor-negative strains of Escherichia coli at low temperature. Therefore, it is concluded that gene 40 product is absolutely required for T4 head assembly only at high temperature. A new location for gene 40 on the T4 genetic map has been established. It maps between gene 41 and gene 42, rather than between gene 58–61 and gene 41. Gene 40 is transcribed counterclockwise, which is concluded from mapping and polyacrylamide gel analysis of gene 40 amber mutants. The product of gene 40 is identified as a protein of MW ∼14,000 under quasi-late regulation whose synthesis gradually decreases at late times. Phage production is greatly reduced in the absence of gene 40 product. However, there seems to be no significant delay in phage maturation in the absence of gene 40 product. Polyheads are produced in suppressor-negative strains of E. coli at both 30° and 42° following gene 40 mutant infection.

T7 gene 6 exonuclease has an RNase H activity

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.

Regulation of gene 32 expression during bacteriophage T4 infection of Escherichia coli.

The gene 32 protein of the bacteriophage T4 plays an important role in genetic recombination, DNA repair, and DNA replication; the protein functions in these processes by virtue of a strong binding capacity for single-stranded DNA. During infections of Escherichia coli by bacteriophage carrying amber of temperature-sensitive mutations in gene 32, the altered gene 32 protein (that is, the amber fragment of the missense polypeptide) is synthesized at greatly elevated rates. During infections by phages that are mutant in other genes (and wild type in gene 32), gene 32 expression is coupled to the quantity of single-stranded DNA produced during the infection. The data are consistent with a model in which the gene 32 protein binds preferentially to all available single-stranded DNA. When all available single-stranded DNA is complexed with gene 32 protein, free gene 32 protein represses its own synthesis. The high level expression of altered gene 32 proteins (amber fragments or missense polypeptides) is a direct consequence of the proposed autoregulation.

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail. Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1 ∗, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII ∗ is present in reduced amounts as compared to wild type. The precursor to B1 ∗ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved. The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973 b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).

Host mutant ( tabD)-induced inhibition of bacteriophage T4 late transcription : II. Genetic characterization of mutants

In this paper we show that the tabD mutants, selected with ts553 or tsCB53, and described in the accompanying paper (Coppo et al., 1975): (a) are recessive to tab +; (b) fail to complement each other, and thus map in the same cistron; (c) by their linkage to rif and their dominance relationships with well characterized amber mutations in the Escherichia coli RNA polymerase operon, probably all map in the gene controlling the synthesis of the β′ subunit of the enzyme. We also describe the isolation of a ts +, k D mutant in phage T4 gene 55, used in the selection of a new tabD mutant ( tabD k292). This tab mutant: (a) generates a defective phenotype which differs somewhat from that of the other tabD mutants; (b) complements the other tabD mutants; (c) by its dominance relationship to amber mutants in the RNA polymerase operon, can be assigned to the structural gene coding for the β subunit of the enzyme. A new type of interaction between T4 genes 55 and 45 is also described. The k D properties of ts553 (gene 55) are suppressed at 30 °C, by a temperature-sensitive mutation in gene 45. This type of interaction between missense mutations in genes 45 and 55 apparently occurs even in tab + strains, since temperature-sensitive mutations in gene 45 accumulate in lysates of two gene 55 mutants ( ts553 and tsA81).

Packaging and maturation of DNA of bacteriophage T7 in vitro.

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phage T7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and lysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 30 degrees . Positive complementation results in a 100-fold increase in phage titer. Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9(-), 10(-)), within head structures present in an extract that contains no phage DNA (gene 5(-)). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5(-), 9(-), 10(-)), which complements gene 19(-)-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.

Maturation of the head of bacteriophage T4: II. Head-related, aberrant τ-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin. The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P23∗, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P23∗. In either case, the DNA-associated particles contain predominantly the cleaved proteins P23∗ and IP III∗, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur. The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.

Requirement of a functional gene 32 product of bacteriophage T4 in UV, repair.

A temperature-sensitive mutation in gene 32 was used to study the role of gene 32 protein in the repair of UV-damaged DNA of bacteriophage T4. It was possible to distinguish between repair and replication of DNA at 33 C. At this temperature, DNA replication continued, and the intracellular DNA was stable. In contrast, no significant repair of UV-damaged DNA was observed even 40 min after the irradiation. Therefore, it was concluded that the defect in the repair mechanism at this temperature is not a simple consequence of the defect in DNA replication but that gene 32 apparently has an independent role for DNA repair. It was reported previously that gene 32 product is required for both T4 DNA replication and genetic recombination. In addition to these findings, this study has given direct evidence that, in vivo, this protein is also essential for the UV repair mechanism.

Gene 6 Exonuclease of Bacteriophage T7: I. PURIFICATION AND PROPERTIES OF THE ENZYME

An exonuclease activity was detectable in crude extracts from Escherichia coli 1200 cells (Endo I-, su-) infected with T7 phage bearing amber mutations in gene 3 (T7 endonuclease I) and gene 5 (T7 DNA polymerase). The activity was not detectable if the infecting phage also contained an amber mutation in gene 6. That gene 6 is the structural gene for this enzyme was shown by the fact that phage bearing a temperature-sensitive mutation in gene 6 induced an exonuclease activity which was more heat labile than the wild type enzyme. The enzyme has been purified 500-fold, and the purified preparations are essentially free of ribonuclease, endonuclease, DNA polymerase, and exonuclease I activity although they do contain measurable 3′-phosphatase activity, likely the reflection of residual E. coli exonuclease III. The enzyme has an absolute requirement for divalent cations and a sulfhydryl reagent and is stimulated greatly by potassium ions. The enzyme shows a marked preference for duplex DNA and liberates 5′-mononucleotides as the sole acid-soluble product. The gene 6 exonuclease is involved in the degradation of cellular DNA to acid-soluble products after T7 phage infection.

The role of gene 49 in DNA replication and head morphogenesis in bacteriophage T4

Mutations in T4 phage genes 33 and 55, genes necessary for the synthesis of late phage-induced proteins, cause the replicating phage DNA to appear in lysates in a form that sediments much more rapidly than the normal 200 s form observed during wild-type infection. We now find that an amber mutation in gene 49 produces this same effect. A temperature-sensitive mutation in gene 49 produces an intermediate DNA form, both at permissive and restrictive temperatubes, indicating that the unusual form of the DNA does not preclude its being converted to phage particles. Mutation in no other late gene tested produces this phenotype. However, we find that inhibition of late protein synthesis with chloramphenicol can partially simulate the effect of gene 33, 55 and 49 mutations. Mutations in gene 49 also prevent the formation of mature 63 s DNA. When the rapidly sedimenting form of replicating phage DNA is isolated and tested as substrate, extracts prepared after T4 infection convert it to slower sedimenting structures. The reaction produces 200 s DNA and apparently terminates with the formation of molecules that sediment between 45 and 65 s. Early-mutant extracts and gene 33-defective and 55-defective extracts do not contain this activity. All late-mutant extracts tested do contain the activity, except those prepared after infection with either of the gene 49 mutants. Suppression of the amber mutation in gene 49 results in a tenfold increase in activity, but the suppressed extracts still possess only 0.1 the wild-type activity. Extracts prepared after ts49 infection also show about 0.1 the wild-type activity, regardless of the temperature of infection or assay. Mixing experiments show that gene 49-defective extracts do not contain an inhibitor of the wild-type activity. Mixed infection with various ratios of 49 - to 49 + phage produces extracts containing activity approximately proportional to the number of 49 + gene copies. Lysates from certain of these mixed infections contain both forms of intracellular DNA and therefore complementation is not as complete as expected of a catalytic function. Gene 49 appears to control a product which, when functional, produces 200 s DNA in lysates of infected cells, but when absent results in the appearance of the more rapidly sedimenting DNA form. This gene product may be an enzyme that functions to alter the structure of the intracellular DNA so that it can be cleaved or encapsulated to form phage particles. Since the activity that we detect in extracts is sensitive to anti-T4 antiserum but not to pre-immune serum, it may be a component of the phage particle itself.