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B Polisky

Publications and source records attributed to B Polisky.

At least 37 records · Page 2Linked to original sources
PubMed1992

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.

Animals↗
PubMed1991

Mutants affecting processing of DNA in macronuclear development in paramecium.

In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type A51 gene, present in micronuclei in two copies and in macronuclei in about 1500 copies. DNA processing, comprised of DNA cleavage, copy number amplification and telomere addition occurs at autogamy and conjugation when old macronuclei degrade and new macronuclei are formed from micronuclei. In this paper we characterize mutants with macronuclear A gene deletions. These mutants are notable in three respects. First, the mutants do not appear to be simple micronuclear deletions. Although genetic analysis shows that the d12 mutant d12(-1300) is homozygous for the allele A-1300 and the mutant d12(+1) for A+1, analysis by the polymerase chain reaction indicates that the micronuclei in these two mutants contain intact, but presumably altered, micronuclear A genes. They undergo deletion during DNA processing when new macronuclei are formed. Second, the position of the deletions in these alleles has been shown to change. The deficiency present in the d12 allele A-1300 was originally determined to extend from position -1300 (relative to the start of translation of the A gene) to the end of the chromosome. Later, a derivative of this strain, homozygous for the d12 allele A+1 was isolated in which the start site of the deletion was found to have moved from -1300 to +1. Third, a surprising interaction occurs in crosses between a line homozygous for the d12 allele and one homozygous for the wild-type A51 allele. Previous work on the non-Mendelian d48 mutant (which has intact A51 genes in its micronucleus, but has truncated A51 genes in its macronucleus) has shown that intact A51 alleles must be present in the old macronucleus in order for A51 alleles to undergo proper processing. We find that d12 alleles act on A51 alleles in heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A51. Thus, in crosses of 51 x d12 (either +1 or -1300) d12 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A51 and truncated d12 alleles. Remarkably the d12 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the A51 allele.

Animals↗
PubMed1991

Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

Animals↗
PubMed1990

Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants.

The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs. One RNA is the primer precursor and the other is a small counter-transcript called RNA I. The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation. We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I. We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription. We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription. During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type. Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Effect of altering GATC sequences in the plasmid ColE1 primer promoter.

Plasmid ColE1 has three recognition sites for the Escherichia coli DNA adenine methylase in the immediate upstream region of the primer promoter. Two of these sites are conserved among all plasmid relatives of ColE1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. Recent experiments have indicated that hemimethylated ColE1-type plasmids are inefficiently replicated after transformation (D. W. Russell and N. Zinder, Cell 50:1071-1079, 1987). By mutating the three methylation sites, we found that disruption of all three GATC sites was necessary for complete relief of the hemimethylation-mediated inhibition of replication in vivo. We also found that these three methylation sites acted in a position-specific manner. The putative cruciform, if present, did not play a regulatory role in the hemimethylation-mediated inhibition of replication.

Base Sequence↗
PubMed1990

Multilevel regulation of surface antigen gene expression in Paramecium tetraurelia.

A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.

Animals↗
PubMed1988

Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro.

We describe three independently isolated copy number mutants of a plasmid ColE1 derivative which undergo temperature- and growth-phase-dependent DNA amplification in Escherichia coli. These mutants have single base-pair alterations in a highly localized region of the plasmid genome encoding the replication primer RNA. The mutations map immediately upstream of the RNA1 transcript, altering the sequence between conserved elements of the RNA1 promoter. These mutants have 2- to 4-fold increased copy number relative to wild-type plasmids in exponential growth at 37 degrees C but undergo 20-fold amplification of copy number relative to wild-type when cells enter stationary phase. Cells containing these plasmids grow with normal kinetics at 37 degrees C but grow poorly at 42 degrees C. The poor growth is associated with high-level plasmid amplification. Both the temperature and growth phase plasmid DNA amplification are suppressed if the ColE1 rop gene product is provided in trans from a compatible plasmid. Analysis of steady-state RNA1 levels indicates that DNA amplification occurs in the presence of RNA1 made by the mutant plasmid. Thus, the DNA amplification of the copy mutant is not due to an inability to synthesize RNA1. Using an in vitro transcription system containing RNase H, we show that mutant primer processing by RNase H is resistant to levels of the replication initiation inhibitor RNA1 that inhibit wild-type primer processing. The defect in inhibition appears not to be at the level of association of RNA1 with nascent primer. These results indicate that mutant plasmid amplification is due to the ability of its primer precursor transcripts to serve as substrates for RNase H despite the presence of RNA1.

Bacteriocin Plasmids↗
PubMed1988

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.

Animals↗
PubMed1987

Suppression of ColE1 RNA-RNA mismatch mutations in vivo by the ColE1 Rop protein.

In the bacterial plasmid ColE1 the control of initiation of DNA replication is mediated by the interaction of two complementary RNA molecules, the replication primer and RNA1. The rate of interaction between RNA1 and the primer RNA in vitro can be increased by the product of the ColE1 rop gene, a 63-amino-acid polypeptide. We have investigated the role of the Rop protein in suppressing the incompatibility defects of 13 RNA1-mutant alleles. These RNA1 mutants are defective due to single nucleotide mismatches with their target, the primer RNA. The rop gene suppresses the defective behavior of most of the RNA1 point mismatch mutants in vivo. However, certain mutations that map in stems I and III of RNA1 are not suppressed by rop. The interaction of wild-type and mutant species of RNA1 with ColE1 replication primer transcripts was studied in vitro in the presence or absence of purified Rop protein. The Rop protein is known to increase the rate of wild-type RNA1-primer interaction about twofold in vitro. This enhancement was also observed for mutant RNA1 species having point alterations or a deletion of the 5' terminus of RNA1, which is consistent with the in vivo suppression results. The implications of these results on the mechanism of Rop activity are considered.

Bacterial Proteins↗
PubMed1987

Transformation of Paramecium by microinjection of a cloned serotype gene.

Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens). A 16-kilobase plasmid containing the gene for immobilization antigen A from Paramecium tetraurelia, stock 51, was injected into the macronucleus of deletion mutant d12, which lacks that gene. Approximately 40% of the injected cells acquired the ability to express serotype A at 34 degrees C. Expression appeared to be regulated normally. The transformed cells, like wild type, could be switched to serotype B by antiserum treatment and culture at 19 degrees C; on transfer to 34 degrees C, they switched back to serotype A expression. Many of the lines retained the ability to express serotype A until autogamy, when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.

Animals↗
PubMed1985

Isolation and characterization of mutants affecting functional domains of ColE1 RNAI.

The control of DNA replication initiation in the plasmid ColE1 is mediated by RNAI, a 108 nucleotide plasmid-encoded RNA that is entirely complementary to the 5'-terminal region of the replication primer RNA. RNAI acts in trans to inhibit primer maturation. Previously, we constructed a plasmid in which the ColE1 RNAI was separated from the primer and placed under transcriptional control of the Serratia marcesens tryptophan promoter. This plasmid provides RNAI in trans in vivo and mediates ColE1-type incompatibility. To determine the critical structural and functional domains of RNAI, we have undertaken a mutational analysis of the RNAI gene carried by this plasmid. We have selected mutants that no longer mediate ColE1-type incompatibility in trans. From the DNA sequences of 18 mutants we have identified mutations at nine new sites in RNAI. In addition, we have determined the secondary structural features of several mutant RNAI species and compared them to wild-type RNAI. Analysis of these mutations has revealed several key features of RNAI secondary structure and function. The domains of RNAI identified in this work which are essential for its function are: the single-stranded loop regions; the integrity of the double-stranded stems; and the single-stranded 5' terminus.

Colicins↗
PubMed1985

Construction of Co1E1 RNA1 mutants and analysis of their function in vivo.

We have carried out experiments designed to investigate the relationship between structure and function for the Co1E1 RNA1 species. RNA1 is a small RNA (108 nucleotides) that has been implicated in copy number control of the multicopy plasmid Co1E1. In vitro, RNA1 inhibits the processing of the primer precursor required for initiation of DNA replication. The RNA1 gene is entirely complementary to the 5'-terminal region of the primer. We have functionally separated these 2 RNA species by cloning the RNA1 gene downstream from the S. marcescens trp promoter. When cloned in a Co1E1-compatible plasmid, a trp-RNA1 fusion has been shown to mediate Co1E1-type incompatibility in vivo. The construction scheme described here also generates mutant RNA1 species with altered sequences at the 5' terminus of RNA1 which have been assayed for function in vivo. These experiments have indicated that sequences at the 5' terminus play a critical role in RNA1 function.

Bacteriocin Plasmids↗
PubMed1985

Alternative conformations of the ColE1 replication primer modulate its interaction with RNA I.

Replication of the ColE1 plasmid is regulated by the interaction of its primer RNA with a small countertranscript (RNA I) that acts as a repressor of functional primer formation. The interaction is dependent on the specific conformations of the complementary RNA molecules. Early in its synthesis, primer adopts an "anti-RNA I" configuration. As transcription proceeds, it is preempted by formation of an alternative domain designated stem-loop IV. This conformational transition has a significant effect on the rate of association of RNA I with the primer in vitro. Nascent primer in the "anti-RNA I" conformation (135 nucleotides) interacts with RNA I 6-fold faster than primer in the stem-loop IV conformation (241 nucleotides), and 35-fold faster than a 567 nucleotide primer precursor. We propose that a conformation-dependent "window of susceptibility" of primer to RNA I exists during primer transcription, and that altered conformations play a role in modulating the rate of functional primer formation.

Bacteriocin Plasmids↗
PubMed1985

Characterization of the ColE1 primer-RNA1 complex: analysis of a domain of ColE1 RNA1 necessary for its interaction with primer RNA.

RNA1 is a small, plasmid-encoded transcript involved in the replication control of the plasmid ColE1. RNA1 blocks replication by preventing processing of the primer RNA necessary for the initiation of replication. It has been proposed that inhibition by RNA1 involves a direct interaction between RNA1 and primer RNA. Here we describe an in vitro system that allows the detection and characterization of the RNA1-primer complex. Using this system, we have demonstrated that the association between ColE1 RNA1 and primer results in the formation of an RNA X RNA hybrid between RNA1 and the 5' end of primer RNA. Furthermore, analysis of mutant RNA1 molecules containing sequence alterations in the 5' single-stranded region of the molecule has revealed that this portion of RNA1 is necessary for the formation of the RNA1-primer complex. These experiments indicate that, in addition to the three single-stranded loops of RNA1, the 5' single-stranded region is a functional domain of the molecule.

DNA Replication↗
PubMed1984

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo.

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens . The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3l nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5'-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5'-terminal region of RNA1 is crucial for its function.

Bacteriocin Plasmids↗
PubMed1984

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein.

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.

Animals↗
PubMed1983

Structural analysis of RNA molecules involved in plasmid copy number control.

DNA replication of the plasmid ColE1 and its relatives is controlled mainly by a small plasmid-encoded transcript known as RNA11. We have studied the conformation of RNA1 molecules from two related but compatible plasmids, ColE1 and RSF1030, using T1 ribonuclease, S1 nuclease, cobra venom nuclease, and diethyl pyrocarbonate modification as probes for secondary structure. Both RNA1 molecules contain three double-stranded stems, three single-stranded loops, and an exposed 5' tail. All loops in both molecules show similar sensitivities to the probes used, as do stems 1 and 3. The region comprising stem 2 of each RNA1 molecule contains the most sequence differences as well as the most structural changes of any region in the two molecules. The structure of the RNA1 molecule encoded by a recessive high copy number mutant of ColE1 was also investigated. Structural alterations involving the first stem and loop of the molecule are proposed to be responsible for the inability of the mutant RNA1 to function in vivo.

Base Sequence↗