bluuebunny/crossref_metadata_2025 huggingface dataset:
p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene.
Authors: Matsuoka S , Edwards M C , Bai C , Parker S , Zhang P , Baldini A , Harper J W , Elledge S J
Published: June 2007

Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.
Cloning and characterization of a TFIIIC2 subunit (TFIIIC beta) whose presence correlates with activation of RNA polymerase III-mediated transcription by adenovirus E1A expression and serum factors.
Authors: Sinn E , Wang Z , Kovelman R , Roeder R G
Published: June 2007

TFIIIC2 is a general factor essential for transcription of 5S RNA, tRNA, and VA RNA genes by mammalian RNA polymerase III and consists of two forms designated TFIIIC2a and TFIIIC2b. TFIIIC2a and TFIIIC2b share common subunits of 220, 102, 90, and 63 kD but differ with respect to transcription activity and the presence of a presumptive 110-kD subunit in the active form (TFIIIC2a). Because both forms can bind the promoter directly, a selective role for the 110-kD subunit in the regulation of RNA polymerase III activity has been suggested. To investigate this possibility, we have cloned and expressed a cDNA encoding the 110-kD subunit (TFIIIC beta). Immunoprecipitation studies with anti-TFIIIC beta antibodies have confirmed that TFIIIC beta is a bona fide subunit present only in TFIIIC2a, that TFIIIC2a and the general factor TFIIIC1 are associated in unfractionated extracts, and that previously undetected polypeptides (potential TFIIIC1 subunits) can be isolated in association with TFIIIC2a. Previous studies have shown that increases in RNA polymerase III activity during infection of cells by adenovirus (with concomitant E1A expression) or during cell growth at high serum concentration results from an increased activity in the TFIIIC fraction. Studies with antibodies to TFIIIC beta have shown that this is strongly correlated with a selective increase in the cellular concentration of the TFIIIC beta 110-kD subunit and a concomitant rise in the ratio of the active-to-inactive forms of TFIIIC2.
The Rhizobium meliloti groELc locus is required for regulation of early nod genes by the transcription activator NodD.
Authors: Ogawa J , Long S R
Published: June 2007

The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. They are required for cell viability but are probably more important for some processes than for others. Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene. The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix-. Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rm1021 megaplasmid pSyma. Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R. meliloti genome. Clones of groESLc and groESLa can each restore robust phage lambda growth on an Escherichia coli groE mutant. Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled. We determined that groELc is required for normal DNA binding of the NodD target sequence in R. meliloti. GroEL coimmunopurifies with NodD1 from R. meliloti, which suggests a direct physical association between these proteins. GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD.
Tissue-specific and allele-specific replication timing control in the imprinted human Prader-Willi syndrome region.
Authors: Gunaratne P H , Nakao M , Ledbetter D H , Sutcliffe J S , Chinault A C
Published: June 2007

To examine the relationship between replication timing and differential gene transcription in tissue-specific and imprinted settings we have studied the replication timing properties of the human Prader-Willi syndrome (PWS) region on human chromosome 15q11-13. Interphase fluorescence in situ hybridization with an overlapping series of cosmid clones was used to map a PWS replication timing domain to a 500- to 650-kb region that includes the SNRPN gene. This PWS domain replicates late in lymphocytes but predominantly early in neuroblasts, with replication asynchrony observed in both tissues, and appears to colocalize with a genetically imprinted transcription domain showing prominent expression in the brain. A 5- to 30-kb deletion in the 5' region of SNRPN results in the loss of late replication control of this domain in lymphocytes when the deleted chromosome is inherited paternally. This potential allele-specific replication timing control region also appears to colocalize with a putative imprinting control region that has been shown previously to abolish the expression of three imprinted transcripts in this same region.
Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members.
Authors: Vairo G , Livingston D M , Ginsberg D
Published: June 2007

Little is known of the mechanisms controlling the G0/G1 transition of the cell cycle. The induction of immediate early gene expression, thought to be important for this process, suggests that the key factors controlling this transition preexist in quiescent cells. The E2F family of transcription factors likely play an important role in this process, because E2F DNA-binding activity exists in quiescent cells, and the induction of at least some immediate early genes requires intact E2F regulatory promoter sites. Here, we show that the major G0 E2F activity of primary human T cells, E2F-4, is stably bound to the p130 pocket protein in association with a DP heterodimerization partner. p130-E2F-4 binding has functional implications because p130 effectively suppressed E2F-4-mediated trans-activation, and coexpression of E2F4 overcame p130-mediated G1 arrest more efficiently than RB-induced G1 blockade. Conversely, E2F-1 overrode an RB-induced G1 block more efficiently than E2F-4. Thus, p130 and RB appear to induce cell cycle arrest via biochemically distinct mechanisms that involve different E2F family members.
Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability.
Authors: Donehower L A , Godley L A , Aldaz C M , Pyle R , Shi Y P , Pinkel D , Gray J , Bradley A , Medina D , Varmus H E
Published: June 2007

By crossing mice that carry a null allele of p53 with transgenic mice that develop mammary adenocarcinomas under the influence of a Wnt-1 transgene, we have studied the consequences of p53 deficiency in mammary gland neoplasia. In Wnt-1 transgenic mice homozygous for the p53 null allele, tumors appear at an earlier age than in animals heterozygous or wild-type at the p53 locus. About half of the tumors arising in p53 heterozygotes exhibit loss of the normal p53 allele, implying selection for p53-deficient cells. Mammary tumors lacking p53 display less fibrotic histopathology and increased genomic instability with aneuploidy, amplifications, and deletions, as detected by karyotype analysis and comparative genomic hybridization. In one tumor, the amplified region of chromosome 7 had an ectopically expressed int-2/FGF3 proto-oncogene, a gene known to cooperate with Wnt-1 in the production of mammary tumors. These findings favor a model in which p53 deficiency relaxes normal restraints on chromosomal number and organization during tumorigenesis.
Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor.
Authors: Naya F J , Stellrecht C M , Tsai M J
Published: June 2007

The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.
Localization of RecA-like recombination proteins on chromosomes of the lily at various meiotic stages.
Authors: Terasawa M , Shinohara A , Hotta Y , Ogawa H , Ogawa T
Published: June 2007

The Rad51 and Lim15 proteins of lily, which are homologs of the bacterial RecA protein, were found on chromosomes in various stages of meiotic prophase 1. The presence of both Rad51 and Lim15 proteins as discrete foci on leptotene and zygotene chromosomes and their colocalization suggest that meiotic recombination begins at the leptotene stage with the cooperation of these proteins and continues in zygotene. Localization of the foci on or adjacent to the chromosomes suggests that these proteins bind to the chromatin loops that extend from the axial cores. The proteins in these foci may participate in the searching and pairing of homologous DNA sequences, as the RecA protein does. The different pattern of localization of the Rad51 protein between the leptotene and pachytene stages and the absence of the Lim15 protein in the pachytene stage suggest that the Rad51 protein plays different roles in these stages.
p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage.
Authors: Macleod K F , Sherry N , Hannon G , Beach D , Tokino T , Kinzler K , Vogelstein B , Jacks T
Published: June 2007

Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to gamma irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL) cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels. p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation.
Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation.
Authors: Udo H , Munoz-Dorado J , Inouye M , Inouye S
Published: June 2007

A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.
A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).
Authors: Steimle V , Durand B , Barras E , Zufferey M , Hadam M R , Mach B , Reith W
Published: June 2007

Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.
The Caenorhabditis elegans spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins kelch and scruin.
Authors: Varkey J P , Muhlrad P J , Minniti A N , Do B , Ward S
Published: June 2007

Six independent mutations in the Caenorhabditis elegans spe-26 gene cause sterility in males and hermaphrodites by disrupting spermatogenesis. Spermatocytes in mutants with the most severe alleles fail to complete meiosis and do not form haploid spermatids. Instead, these spermatocytes arrest with missegregated chromosomes and mislocalized actin filaments, endoplasmic reticulum and ribosomes. In spite of this arrest some of the nuclei and the organelles that normally transport sperm-specific components to the spermatid mature as if they were in spermatids. The spe-26 gene is expressed throughout the testis in both spermatogonial cells and spermatocytes. It encodes a 570-amino-acid polypeptide, which contains five tandem repeat motifs, each of approximately 50 amino acids. These repeats are similar in sequence to repeats in the Drosophila kelch protein, in the invertebrate sperm protein scruin that cross-links actin filaments, as well as in the mouse and pox virus proteins. The functional importance of these repeat motifs is shown by the fact that five of the spe-26 mutations are in the tandem repeats, and one of the most severe mutations is a substitution in a highly conserved glycine. These results suggest that spe-26 encodes a cytoskeletal protein, perhaps actin binding, which is necessary to segregate the cellular components that form haploid spermatids.
The dishevelled protein is modified by wingless signaling in Drosophila.
Authors: Yanagawa S , van Leeuwen F , Wodarz A , Klingensmith J , Nusse R
Published: June 2007

Wingless (Wg) is an important signaling molecule in the development of Drosophila, but little is known about its signal transduction pathway. Genetic evidence indicates that another segment polarity gene, dishevelled (dsh) is required for Wg signaling. We have recently developed a cell culture system for Wg protein activity, and using this in vitro system as well as intact Drosophila embryos, we have analyzed biochemical changes in the Dsh protein as a consequence of Wg signaling. We find that Dsh is a phosphoprotein, normally present in the cytoplasm. Wg signaling generates a hyperphosphorylated form of Dsh, which is associated with a membrane fraction. Overexpressed Dsh becomes hyperphosphorylated in the absence of extracellular Wg and increases levels of the Armadillo protein, thereby mimicking the Wg signal. A deletional analysis of Dsh identifies several conserved domains essential for activity, among which is a so-called GLGF/DHR motif. We conclude that dsh, a highly conserved gene, is not merely a permissive factor in Wg signaling but encodes a novel signal transduction molecule, which may function between the Wg receptor and more downstream signaling molecules.
A multidrug resistance transporter/serine protease gene is required for prestalk specialization in Dictyostelium.
Authors: Shaulsky G , Kuspa A , Loomis W F
Published: June 2007

The prestalk-specific gene, tagB, was disrupted by restriction enzyme-mediated integration (REMI) mutagenesis. Mutant aggregates exhibit a cell-autonomous defect in specialization of PST-A cells, a prestalk subpopulation that forms the tip and eventually forms the stalk of the fruiting body. Cooperative (non-cell-autonomous) defects were found in sporulation and in specialization of prestalk cells that eventually form the upper cup of the fruiting body (PST-O). The pattern of ecmA::lacZ expression in mutant tagB- cells defines a primary prestalk population, PST-I, from which other prestalk cells differentiate. After PST-A cells differentiate, they induce remaining PST-I cells to become PST-O cells. Subsequently, prestalk cells induce encapsulation of prespore cells during culmination. tagB is homologous to serine protease and to multidrug resistance (MDR) transporter genes, implying a mechanism of action that includes proteolysis and export of peptide signals. Intercellular communication via TagB may mediate integration of cellular differentiation with morphogenesis.
Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons.
Authors: Signon L , Kleckner N
Published: June 2007

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.
Enhancer-trap targeting at the Broad-Complex locus of Drosophila melanogaster.
Authors: Gonzy-Tréboul G , Lepesant J A , Deutsch J
Published: June 2007

Here, we describe the exact replacement of a defective unmarked P element by an enhancer-trap transposon marked by the miniwhite gene and carrying lacZ as a reporter gene. The original defective P element was located in an intron of the Broad-Complex (BRC), a key gene involved in metamorphosis. Replacement events resulted from conversions induced by the P-element transposase from a donor enhancer-trap element located on another chromosome. Six independent conversion events were selected. In all converted chromosomes, the enhancer-trap transposon was in the same orientation as the original P element. From the pattern of X-gal staining observed, lacZ expression likely reflects the regulatory influence of BRC enhancers on the convertant transposon. Reversion to wild type was achieved by excision of the enhancer-trap transposon. The six convertants were analyzed in detail at the nucleotide level. The occurrence of a polymorphism at position 33 of the P-element sequences led us to propose a conversion mechanism involving homologous P sequences for repair. This is in contrast to previously analyzed P-element transposase-induced conversion events and proposed models relying on sequence identity between genomic Drosophila sequences. The lack of any homology requirement other than between P element sequences means that our findings can be easily generalized. Targeting a marked P-element derivative at a precise site without loss or addition of genetic information makes it possible to exploit the hundreds of defective P elements scattered throughout the Drosophila genome by replacing them with engineered P elements, already available.
PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.
Authors: Greer C E , Lund J K , Manos M M
Published: June 2007

The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.
The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria.
Authors: Barry T , Colleran G , Glennon M , Dunican L K , Gannon F
Published: June 2007

Variable regions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms. In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms. This study presents a general method to obtain species-specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes. The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes. Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacteria. Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest. In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail. A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus.
Degenerate oligonucleotide sequence-directed cross-species PCR cloning of the BCP 54/ALDH 3 cDNA: priming from inverted repeats and formation of tandem primer arrays.
Authors: Cooper D L , Baptist E W
Published: June 2007

Bovine corneal protein 54 (BCP 54) is the major soluble protein of the bovine cornea, and immunoreactive forms of this protein have been described in a wide range of mammals. Dideoxy sequence determination of a previously synthesized 420-bp cDNA to BCP 54 generated by the novel mixed oligonucleotide primer amplification of cDNA (MOPAC) procedure revealed extensive similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). PCR amplification with additional pairs of degenerate oligonucleotide sequence (DOS) primers derived from both BCP 54-amino-acid sequence and amino acid and nucleotide sequence data from RATALD produced three PCR products that were cloned and subsequently sequenced. The major product was 716-bp BCP 54 cDNA clone encompassing the BCP 54 carboxy-terminal amino acid sequence for which the DOS pair was designed. Sequence alignment of the BCP 54 cDNA and its translation product with RATALD demonstrated 81% and 85% identity at the nucleotide and amino acid levels, respectively. Analysis of the additional two clones established that they were the results of PCR artifactual processes. The first of these was a 552-bp product occurring at elevated primer concentrations that formed through bidirectional amplification from a single DOS annealing to an inverted repeat located in the BCP 54 coding sequence. The second artifactual product was a 212-bp sequence that contained several unreported amplification anomalies, including the formation of a tandem primer array.
RAPD and other PCR-based analyses of plant genomes using DNA extracted from small leaf disks.
Authors: Deragon J M , Landry B S
Published: June 2007

A nondestructive, early DNA diagnostic system to implement marker-assisted selection in plant breeding programs has been developed. The main components of the system are a rapid and simple DNA microextraction method and fast DNA polymorphism analyses based on site-specific or arbitrary DNA amplification. A small disk (5 mm diameter) is collected from one cotyledon or the first leaf of a young seedling using a common paper punch. Disruption of plant tissues is done by enzymatic digestion of cell walls. This ensures protection from sample-to-sample contamination and uniform DNA yield. DNA isolated from the resulting protoplasts is sufficient to perform a minimum of five and a maximum of 20 PCR reactions/sample. Total DNA, nuclear DNA, and RNA can be analyzed selectively. The system has been tested successfully with eight major crops. Amplification products generated with DNA prepared with this quick procedure are equivalent to those obtained from CsCl-purified DNA. Up to 120 plants can be treated in 2 days and the procedure lends itself to automation. Potential applications in plant breeding will be discussed.
Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR.
Authors: Subramanian P S , Versalovic J , McCabe E R , Lupski J R
Published: June 2007

We describe a novel method to map chromosomal Escherichia coli::Tn5 insertion mutations rapidly. This method utilizes the ends of Tn5 and the E. coli REP sequence as primer binding sites for the polymerase chain reaction (PCR). The unique E. coli chromosomal sequence located between these primer binding sites is amplified by PCR and used as a probe to identify the recombinant clones from the Kohara phage ordered E. coli miniset bank that contains the Tn5 mutated loci. We used this approach to map two Tn5 insertion mutations previously identified by their effect on glycerol metabolism. The insertion mutations mapped to glpD, the aerobic sn-glycerol-3-phosphate dehydrogenase gene. Phenotypic analysis of the mutant strains revealed one with partial GlpD activity, suggesting transposon-mediated alteration of promoter activity. This mapping method should be applicable to the rapid physical mapping of any insertion mutation in the E. coli chromosome.
Sequencing of PCR-amplified DNA.
Authors: Bevan I S , Rapley R , Walker M R
Published: June 2007

Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the use of the PCR product for other purposes either prior to or subsequent to generation of sequence data. The single-stranded sequencing methods generally require some prior decision regarding sequencing of the product. Assisted by automated workstation development, sequencing of single-stranded DNA PCR products generated either during thermal cycling or following affinity-capture strand separation may have significant future utility, particularly in genome mapping and routine clinical diagnosis. Despite template type and protocol differences, in all situations the purity and concentration of PCR-amplified DNA template used remains the most critical factor determining the efficiency and reliability of nucleotide sequencing methods.
Preferential PCR amplification of alleles: mechanisms and solutions.
Authors: Walsh P S , Erlich H A , Higuchi R
Published: June 2007

The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed.
Simplified template preparation and improved direct sequencing using Taq polymerase.
Authors: Srivastava A K , Montanaro V , Kere J
Published: June 2007

A streamlined version of direct dideoxy sequencing is presented that includes template preparation as well as sequencing protocols. The method is used routinely to sequence double-stranded PCR products after minimal purification with one of the primers used in amplification. Either 35S or 32P labeling can be used with equally good results.
Rapid synthesis of DNA deletion constructs for mRNA quantitation: analysis of astrocyte mRNAs.
Authors: Galea E , Feinstein D L
Published: June 2007

A rapid method for the synthesis of DNA fragments for competitive PCR analysis is described. This procedure takes advantage of the fact that if PCR is carried out with a mixture of re-ligated restriction digestion fragments, only those fragments containing binding sites for both PCR primers will be amplified. Following electrophoresis of the amplified mixture, the DNA fragment of desired size can be excised from an agarose gel, reamplified, and used for subsequent competitive PCR. We have used this procedure to synthesize deletion constructs for the rat glial fibrillary acidic protein (GFAP) gene, and have used competitive PCR to determine the levels of this mRNA in primary cultures of rat brain astrocytes.
Amplification and cloning of sugarcane sucrose synthase cDNA by anchored PCR.
Authors: Kumar A S , Moore P H , Maretzki A
Published: June 2007

We have used a strategy based on the polymerase chain reaction (PCR) to amplify and construct full-length sucrose synthase (SS) cDNA of sugarcane. Two SS-specific internal primers were synthesized based on their complementarity to published consensus sequences of the SS gene of maize and wheat. Amplification of full-length cDNA was achieved by an anchored PCR method utilizing primers which extend to 5' and 3' ends of specific cDNA. In the first step, a homopolymeric oligo(dC) tail was added to the 3' end of single-stranded cDNAs. The two SS cDNAs were amplified, one with a 5' end (SSp1) and the other with a 3' end (SSp2) using one internal SS primer and the other anchored end primer. Finally, overlapping fragments were identified by restriction mapping, and the non-overlapping fragments were excised and religated to reconstruct full-length cDNA. Partial sequences of the reconstructed cDNAs (SS-5' and SS-3') were compared with the published SS sequences to confirm that the amplified DNA was a copy of the SS transcript.
Modeling of heteroduplex formation during PCR from mixtures of DNA templates.
Authors: Ruano G , Kidd K K
Published: June 2007

We have investigated the capability of PCR to amplify a specific locus from each template in a mixture of allelic DNAs. Modeling experiments employed a 300-bp HOX2B segment as target and utilized, as distinct template "alleles," genomic DNAs from 1 human and from 2 chimpanzees known to differ in sequence at the target. Two modes were used: (1) mixtures of PCR products that had been previously amplified individually from a single template and (2) PCR amplification en masse from composite human/chimpanzee genomic DNA templates. Products generated by either mode were separated by denaturing gradient gel electrophoresis (DGGE). Detection of a "trace" allele mixed with a "dominant" one was possible by simple ethidium bromide staining of the gel up to a sensitivity of 1 part in 20. A balanced mixture was represented by 5:5 and 4:3:3 mixtures of allelic PCR products or genomic templates; an uneven mixture of dominant and trace alleles, by 9:1 and 8:1:1 mixtures. For 5:5, two homoduplex bands and two heteroduplex bands of equal intensity were generated. For 9:1, the trace homoduplex disappears whereas the two heteroduplexes are easily visible. For 8:1:1, four heteroduplexes and one homoduplex were observed; homoduplexes and heteroduplexes formed from the trace alleles were not visible. These experiments demonstrate that PCR can amplify mixed allelic templates in direct proportion to the stoichiometric fraction of each template. Trace species are captured as heteroduplexes with the most abundant species and are clearly displaced on denaturing gradient gels from the dominant homoduplex species. Our analytical studies can be applied to analysis of sequence variants in DNA collected from cancerous or infected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
In situ localization of PCR-amplified human and viral cDNAs.
Authors: Nuovo G J , Gorgone G A , MacConnell P , Margiotta M , Gorevic P D
Published: June 2007

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.
Improved blood sample processing for PCR.
Authors: Casareale D , Pottathil R , Diaco R
Published: June 2007

To simplify our procedure for blood sample processing for PCR, we introduced a simpler, shorter, and more cost-effective method for the separation of peripheral blood lymphocytes (PBL) and DNA extraction for amplification with Taq polymerase. By this method, blood samples are processed in two simple 15-min steps: (1) separation of PBLs from whole blood by red blood cell lysis with the Roche Specimen Washing Solution, and (2) DNA extraction by heat-detergent treatment of separated PBLs. This new method is simpler than the standard Ficoll-Hypaque method for PBLs separation and Proteinase K digestion for DNA extraction. It is not inhibitory to DNA amplification and it allows effective processing of blood samples even after prolonged storage (as long as 8 days) at room temperature.
Detection of mutation delta F508 in the cystic fibrosis gene using allele-specific PCR primers and time-resolved fluorometry.
Authors: Iitiä A , Høgdall E , Dahlen P , Hurskainen P , Vuust J , Siitari H
Published: June 2007

A method to detect the main cystic fibrosis (CF) mutation delta F508 from dried blood spots, whole blood, or saliva using the polymerase chain reaction (PCR) and time-resolved fluorometry (TRF) is described. Samples are treated by boiling in mild alkaline solution, after which two allele-specific PCR reactions are performed. Allele-specific primers and a common biotinylated primer are used in the amplification reactions. To detect the PCR product, an europium-labeled oligonucleotide, complementary to the biotinylated strand of the PCR product, is used in a solution hybridization. Hybridization is done in streptavidin-coated microtitration wells, making the detection easy to perform. After a washing step, the bound label is detected using a time-resolved fluorometer. To analyze function of the assay, 20 dried blood spot samples were tested. PCR amplification of the deletion region combined with gel retardation assay was used as a control method. In the initial testing, 2 samples giving discrepant results in the two assays were found. In addition, 17 samples from known CF patients together with 6 normal control samples were analyzed. Among these patient samples, 10 homozygotes and 6 carriers for mutation delta F508 were found.
Immunomagnetic recovery of Chlamydia trachomatis from urine with subsequent colorimetric DNA detection.
Authors: Hedrum A , Lundeberg J , Påhlson C , Uhlén M
Published: June 2007

There is need for integrated systems that provide rapid and sensitive diagnosis of Chlamydia trachomatis. Here we describe for the first time that specific antibodies to a C. trachomatis surface protein can be used for magnetic recovery of bacteria directly from crude urine samples. The combination of immunomagnetic separation (IMS), where the bacterial cells are both purified and concentrated, with a colorimetric PCR assay makes the method sensitive and suitable for routine clinical screening in microtiter format. No filtrations, centrifugations, extractions, or electrophoretic techniques are required throughout the whole procedure. Using magnetic beads as the solid phase, the colorimetric detection of immobilized amplified nucleic acids (DIANA) also allow direct DNA sequencing of positive samples for more detailed analysis. Of the 27 urine samples analyzed, eight were found to be positive using this method. Only five out of these eight were determined to be positive by cell culture.
Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element.
Authors: Chiang L W , Kovari I , Howe M M
Published: June 2007

Saturation mutagenesis is one approach for determining the contributions of individual base pairs to the structure and function of defined DNA sequence elements. In this paper, we describe a novel method for saturation mutagenesis involving PCR amplification with degenerate synthetic oligonucleotides as primers. The degeneracy is confined to a specific target within the primer by mixing a low percentage of the three non-wild type (non-WT) nucleotide precursors with WT at specific positions during primer synthesis. PCR amplification of WT template DNA with the degenerate primer and an opposing WT primer, followed by subsequent cloning using restriction sites designed into the primers, results in recovery of a population of randomly mutated products. Since primers with multiple mutations hybridize less efficiently to WT template DNA during PCR amplification, the recovery of mutants with multiple base changes is greatly reduced. The efficient generation of random point mutations with this method allows the construction of separate mutant populations, each mutagenized over a different portion of the DNA sequence element. If a phenotypic assay is available, these populations can be screened directly to define those regions within the element that are important for activity. Only those populations containing mutations in the important regions require further characterization by DNA sequence analysis.
Synthesis and characterization of 5'-fluorescent-dye-labeled oligonucleotides.
Authors: Giusti W G , Adriano T
Published: June 2007

Fluorescent-dye-labeled oligonucleotides are used in many procedures, including DNA sequencing, PCR, restriction mapping, the study of genetic disease, and forensics identification. In this paper, we describe detailed methods for the synthesis, purification, and quantification of 5'-fluorescent-dye-labeled oligonucleotides. The relationship of specific and nonspecific dye attachment to synthetic oligonucleotides is discussed, and the importance of primer design is considered as a way to prevent chemical problems from interfering with biological reactions.