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S Casjens

Publications and source records attributed to S Casjens.

At least 19 recordsLinked to original sources
PubMed1995

Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order.

We have constructed physical and genetic maps of the chromosomes of 21 Lyme disease agent spirochetes from geographically diverse locations. All have linear chromosomes whose lengths range from 935 to 955 kbp, and all contain multiple linear plasmids in the 16- to 175-kbp size range. The locations of 11 gene clusters on the chromosomes of these different isolates are indistinguishable at the resolution achieved in this study, indicating that the members of this related group of species have highly conserved chromosomal gene orders. However, chromosomal restriction endonuclease cleavage site maps are unique for nearly all isolates. The 22 chromosomal maps currently available define eight classes of Lyme disease agents. Four of these correspond to the previously proposed species Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, and Borrelia japonica. In addition, the North American isolates 21038, DN127 c19-2, 25015, and CA55 typify four additional chromosomal types that are as phylogenetically distinct as the species listed above. These findings support the idea that comparison of restriction maps is currently the most robust and definitive method for determining overall chromosomal relationships among closely related bacteria. In the course of this work, we located on the chromosome the previously unmapped outer surface protein-encoding LA7 gene and genes homologous to the Escherichia coli priA, plsC, parE, and parC genes, and we have substantially refined the locations of the recA, fla, p22A, and flgE genes.

Animals↗
PubMed1994

Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins.

The in vivo conformational substrates of the GroE chaperonins have been difficult to identify, in part because of limited information on in vivo polypeptide chain folding pathways. Temperature-sensitive folding (tsf) mutants have been characterized for the coat protein and tailspike protein of phage P22. These mutations block intracellular folding at restrictive temperature by increasing the lability of folding intermediates without impairing the stability or function of the native state. Overexpression of GroEL/ES suppressed the defects of tsf mutants at 17 sites in the coat protein, by improving folding efficiency rather than assembly efficiency or protein stability. Immunoprecipitation experiments demonstrated that GroEL interacted transiently with newly synthesized wild-type coat protein and that this interaction was prolonged by the tsf mutations. Folding defects of the tailspike polypeptide chains were not suppressed. A fraction of the tsf mutant tailspike chains bound to GroEL but were inefficiently discharged. The results suggest that 1) thermolabile folding intermediates are natural substrates of GroEL/ES; 2) although GroEL may bind such intermediates for many proteins, the chaperoning function is limited to a subset of substrate proteins; and 3) a key reason for the heat-shock response may be to stabilize thermolabile folding intermediates at elevated temperatures.

Bacteriophage P22↗
PubMed1993

Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent.

A physical map of the 952 kbp chromosome of Borrelia burgdorferi Sh-2-82 has been constructed. Eighty-three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96 kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh-2-82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.

Blotting, Southern↗
PubMed1992

Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.

Bacteriophage P22↗
PubMed1992

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.

Base Sequence↗
PubMed1991

Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging.

The mechanism of DNA packaging by dsDNA viruses is not well understood in any system. In bacteriophage P22 only five genes are required for successful condensation of DNA within the capsid. The products of three of these genes, the portal, scaffolding, and coat proteins, are structural components of the precursor particle, and two, the products of genes 2 and 3, are not. The scaffolding protein is lost from the structure during packaging, and only the portal and coat proteins are present in the mature virus particle. These five genes map in a contiguous cluster at the left end of the P22 genetic map. Three additional genes, 4, 10, and 26, are required for stabilizing of the condensed DNA within the capsid. In this report we present the nucleotide sequence of 7461 bp of P22 DNA that contains the five genes required for DNA condensation, as well as a nonessential open reading frame (ORF109), gene 4, and a portion of gene 10. N-terminal amino acid sequencing of the encoded proteins accurately located the translation starts of six genes in the sequence. Despite the fact that most of these proteins have striking analogs in the other dsDNA bacteriophage groups, which perform highly analogous functions, no amino acid sequence similarity between these analogous proteins has been found, indicating either that they diverged a very long time ago or that they are the products of spectacular convergent evolution.

Amino Acid Sequence↗
PubMed1991

Fine structure genetic and physical map of the gene 3 to 10 region of the bacteriophage P22 chromosome.

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes.

Chromosome Mapping↗
PubMed1989

Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis.

The nucleotide sequence of a 2558-bp region of bacteriophage P22 at the right end of the genetic map between genes 19 and 3 was determined. A new gene that is partially required for lytic growth, named gene 15, was noted. P22 mutants were constructed which lack gene 15 function, and the gene 15 product was found to be required for lysis in the presence of some divalent cations. It has extensive amino acid sequence similarity with the phage lambda Rz gene, which has a similar function, and weak similarity to the phage T7 18.5 gene which previously had no known function. A hybrid P22 phage, in which the T7 18.5 gene replaces the P22 gene 15, exhibits the plating properties of wild-type P22, strongly suggesting that the two genes have similar functions. In addition, deletions were constructed which show that phage P22 has no additional genes required for lytic growth of lysogeny between genes 19 and 3.

Amino Acid Sequence↗
PubMed1988

A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60.

A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase. This interruption is part of the transcribed messenger RNA and appears not to be removed before translation. Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case. The interruption is bracketed by a direct repeat of five base pairs. A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation. The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.

Amino Acid Sequence↗
PubMed1988

Analysis in vivo of the bacteriophage P22 headful nuclease.

Bacteriophage P22 packages its double-stranded DNA chromosomes from concatemeric replicating DNA in a processive, sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally (rightward) from that point. DNA ends are generated near the pac site before or during the condensation reaction. The right end of the mature chromosome is created by a cut made in the DNA by the "headful nuclease" after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here accurate measurements of the P22 chromosome length (43,400( +/- 750) base-pairs, where the uncertainty is the range in observed lengths), genome length (41,830( +/- 315) base-pairs, where the uncertainty represents the accuracy with which the length is known), the terminal redundancy (1600( +/- 750) base-pairs or 3.8( +/- 1.8)%, where the uncertainty is the observed range) and the imprecision in the headful measuring device ( +/- 750 base-pairs or +/- 1.7%). In addition, we present evidence for a weak nucleotide sequence specificity in the headful nuclease. These findings lend further support to, and extend our understanding of, the sequential series model of P22 DNA packaging.

DNA, Viral↗
PubMed1987

Initiation of bacteriophage P22 DNA packaging series. Analysis of a mutant that alters the DNA target specificity of the packaging apparatus.

Bacteriophage P22 is thought to package its double-stranded DNA chromosome from concatemeric replicating DNA in a "processive" sequential fashion. According to this model, during the initial packaging event in such a series the packaging apparatus recognizes a nucleotide sequence, called pac, on the DNA, and then condenses DNA within the coat protein shell unidirectionally from that point. DNA ends are generated near the pac site before or during the condensation reaction. The opposite end of the mature chromosome is created by a cut made in the DNA after a complete chromosome is condensed within the phage head. Subsequent packaging events on that concatemeric DNA begin at the end generated by the headful cut of the previous event and proceed in the same direction as the previous event. We report here the identification of a consensus nucleotide sequence for the pac site, and present evidence that supports the idea that the gene 3 protein is a central participant in this recognition event. In addition, we tentatively locate the portion of the gene 3 protein that contacts the pac site during the initiation of packaging.

Base Sequence↗
PubMed1986

Plasmid vectors useful in the study of translation initiation signals.

The construction and characterization of plasmid vectors useful in the analysis of translation initiation signals in Escherichia coli and in the construction of lacZ gene hybrids are described. Transcription on the vectors proceeds from a cAMP-independent lac promoter through several restriction sites into a truncated lacZ structural gene lacking its first eight codons. Because this gene has no translation initiation signal, its level of expression is extremely low. A DNA fragment containing a translation start signal can be inserted between the promoter and truncated lacZ gene to produce a hybrid protein with functional beta-galactosidase activity. The vectors described here differ in sequence between the EcoRI cloning site and the lacZ gene to allow easy, in-frame joining of DNA containing a translation initiation signal to the lacZ gene. Cells containing plasmids can be screened directly for in-frame inserts by colony color on indicator plates.

Genetic Vectors↗
PubMed1985

Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits.

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.

Adenosine Triphosphate↗
PubMed1985

Bacteriophage L: chromosome physical map and structural proteins.

Restriction endonuclease cleavage site mapping was used to locate the regions of highest sequence homology in the chromosomes of Salmonella typhimurium bacteriophages L and P22. These lie in the DNA packaging, tail, early transcription antitermination, and perhaps integration "gene modules." Other regions of the two genomes are substantially less closely related. Phage L, which has no functional immunity I region, lacks approximately 1300 bp of DNA when compared to P22 in this section of the chromosome. At least some of the virion structural proteins are interchangeable between the two phages, which suggests that the two phage structural protein genes are very closely related. In addition, the apparent molecular weights of most P22 and L phage structural proteins are very similar. However, the phage L virion contains about 140 molecules of a 15K capsid protein which apparently has no P22 analog.

Chromosome Mapping↗
PubMed1985

Assembly-controlled autogenous modulation of bacteriophage P22 scaffolding protein gene expression.

In the assembly of bacteriophage P22, precursor particles containing two major proteins, the gene 5 coat protein and the gene 8 scaffolding protein, package the DNA molecule. During the encapsidation reaction all of the scaffolding protein molecules are released intact and subsequently participate in further rounds of DNA encapsidation. We have previously shown that even though it lies in the center of the late region of the genetic map, the scaffolding protein gene is not always expressed coordinately with the remainder of the late proteins and that some feature of the phage assembly process affects its expression. We present here in vivo experiments which show that there is an inverse correlation between the amount of unassembled scaffolding protein and the rate of scaffolding protein synthesis and that long amber fragments of the scaffolding protein can turn down the synthesis of intact scaffolding protein in trans. These results support a model for scaffolding protein regulation in which the feature of the assembly process which modulates the rate of scaffolding protein synthesis is the amount of unassembled scaffolding protein itself.

Capsid↗
PubMed1985

Bacteriophage P22 tail protein gene expression.

We have found that mutations which block bacteriophage P22 head assembly at or before the DNA packaging stage (1-, 2-, 3-, 5-, and 8-) cause up to a 20-fold increase in the amount of tail (gene 9) protein made during infection. This correlation seems strong enough to warrant consideration of a control mechanism in which the failure to package DNA per se causes a large increase in the synthesis of tail protein. Our results indicate that one of the repressors required for maintenance of lysogeny, the mnt gene product, may be partially responsible for this phenomenon.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Posttranscriptional modulation of bacteriophage P22 scaffolding protein gene expression.

The bacteriophage P22 late operon contains 2 genes whose products are required for cell lysis and 13 genes whose products are involved in the morphogenesis of the phage particle. This operon is under the positive control of the phage gene 23 product and is thought to have a single promoter. The expression of one of these late genes, the scaffolding protein gene, is autogenously modulated independently from the remainder of the late genes. When unassembled, scaffolding protein turns down the rate of synthesis of additional scaffolding protein, and when it is assembled into phage precursor structures, it does not. Experiments presented here show (i) that the mRNA from the scaffolding protein gene is functionally threefold more stable when most of the scaffolding protein is assembled than when it is unassembled and (ii) that no new promoter near the scaffolding protein gene is activated at the high level of synthesis. These data support the model that this autogenous modulation occurs at a posttranscriptional level. We also observed that another message, that of coat protein, appears to become increasingly stable with time after phage infection.

Base Composition↗