DNA identification of commercial ginseng samples.

An investigation was performed with the objective of developing a DNA-based protocol for the identification of commercial samples of the herbal compound ginseng. There are currently two major herbal products referred to as ginseng. They are Korean or Chinese ginseng (Panax ginseng) and American ginseng (Panax quinquefolius). The market for ginseng in the United States is estimated to be approximately $300 million annually. Current tests for ginseng species identification rely on expert botanical identification of fresh plant/root specimens or on biochemical characterization of active and marker compounds (e.g., ginsenosides). For the determination of the feasibility of ginseng identification by DNA analysis, a strategy based on the direct DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region was developed. Other genetic tests included sequence analysis of the chloroplast ribulose 1,5-bisphosphate carboxylase large subunit gene and DNA fingerprinting by the rapid amplification of polymorphic DNA technique. To confirm the results, each ginseng sample was identified using high-performance liquid chromatography. All methods were successful in distinguishing American from Korean ginseng. In addition, the protocol was improved for the isolation of genomic and plastid DNA from commercial ginseng preparations by incorporating an impact homogenization step into the standard column chromatography purification procedure.

Recombinant expression of the antimicrobial peptide polyphemusin and its activity against the protozoan oyster pathogen Perkinsus marinus.

Polyphemusin is a broad-spectrum antimicrobial peptide isolated from hemocytes of the North American horseshoe crab Limulus polyphemus. To date the polyphemusin used for scientific analyses has been purified from the natural materials or obtained by chemical synthesis. We report here the recombinant expression in Escherichia coli, and subsequent purification, of a polyphemusin analogue (rLim1). To prevent toxicity of the antimicrobial peptide in the highly susceptible E. coli host, we used a carboxy-terminal fusion protein cloning strategy provided by a maltose-binding protein (MBP) gene fusion system (New England Biolabs). Antimicrobial activity of recombinant polyphemusin was similar to that seen with amidated native polyphemusin peptide. When rLim1 was tested for antibiotic activity against the apicomplexan protozoan oyster pathogen Perkinsus marinus, complete inhibition was observed at 12 micrograms/ml, and partial inhibition at 8 micrograms/ml.

Headful packaging revisited: the packaging of more than one DNA molecule into a bacteriophage P1 head.

Like a variety of other bacteriophages, such as T4 and P22, bacteriophage P1 packages DNA by a "headful" mechanism in which the capacity of the viral capsid determines the size of the single DNA molecule that is packaged. Because of the long-standing and general acceptance of this packaging mechanism, we were surprised to discover that some of our observations, using the in vitro P1 packaging system, could be explained by the packaging of less than headful-sized (< 110 kb) DNA molecules into a P1 capsid. To account for these observations, we describe results that support a model of in vitro P1 packaging in which multiple less than headful-sized DNA molecules are taken into a P1 head until that head has been filled. The results further suggest that the phage so generated can occasionally inject more than one DNA molecule into a cell upon viral infection. The data that supports these conclusions are: (1) the DNAs of the circular P1 cloning vectors pAd10sacBII (32 kb) and pNS358 (14 kb) are packaged in vitro with an efficiency of about 6 to 12% of that of longer concatemers of these DNAs. (2) The in vitro packaging of two differentially marked, less than 18 kb plasmid DNAs in the same reaction results in the production of a phage that can occasionally inject both DNAs into the same cell upon infection. (3) Virus particles generated by the packaging of either pAd10sacBII plasmid DNA or the two differently marked plasmids have a density in CsCl equilibrium gradients that is the same as P1 plaque-forming phage, suggesting that the former phage contain a headful of DNA. These results cannot be explained by Cre-mediated site-specific recombination between plasmids in the P1 packaging extracts. Finally, we present in vivo experiments that are also consistent with the headful packaging of multiple DNAs into a P1 head.

Physical mapping of the rat tissue kallikrein family in two gene clusters by analysis of P1 bacteriophage clones.

In several mammalian species the tissue kallikreins constitute gene families whose members encode a highly related subgroup of the simple serine proteases. Previous characterization of kallikrein genes in the rat identified 13 of the potential 15-20 members present in this species. To characterize comprehensively all members of the rat gene family and to define the linkage of family members, we have isolated clones bearing kallikrein genes from a rat genomic library constructed with the P1 bacteriophage vector pAd10-SacBII. Library pools containing kallikrein genes were identified by polymerase chain reaction with primers complementary to regions highly conserved among members of the rat gene family. Individual members of the family within the library pools were identified with gene-specific PCR assays that rely upon short divergent regions among the family members. Detection of multiple kallikrein family members within single library pools suggested tight linkage of the individual genes. Isolation and analysis of 12 kallikrein P1 clones confirmed the linkage of gene family members and established a physical map for two clusters of genes at the kallikrein locus. The linkage relationships of the known gene family members within the two gene contigs are rKLK1-rKLK3-rKLK7-rKLK9 and rKLK8-rKLK2-rKLK6-rKLK4-rKLK10-rKLK12++ +. Pulsed-field electrophoretic analysis of rat genomic DNA demonstrated linkage between the two gene clusters, which form an extended locus that is most narrowly defined by a 440-kb BssHII fragment, and identified an unmethylated CpG island that is tightly linked to this locus.

In-frame cloning of large synthetic genes using moderate-size oligonucleotides.

A method is described that facilitates the cloning of large synthetic genes in plasmid vectors such as pUC and pGEM. This protocol uses unpurified synthetic oligonucleotides of moderate length (ca. 50-90 bp) to construct larger DNA molecules by a reiterative, directional cloning procedure. Open reading frames are maintained by the flexible use of six-base pair blunt-end restriction sites that are not present in the DNA sequence of the plasmid cloning vector. Rapid production and analysis of plasmid intermediates are achieved by standard recombinant DNA cloning methods. The use of an anchored sticky-end restriction site and a variable blunt-end restriction site in each step of the cloning scheme gives specific orientation to each oligonucleotide fragment and results in high cloning efficiencies. The flexibility of the method allows for the construction of a gene of any size. Very large synthetic genes can be made by generating the intermediate gene fragments in parallel vectors and simply cloning the fragments in frame to produce the final construct.

P1 and cosmid clones define the organization of 280 kb of the mouse H-2 complex containing the Cps-1 and Hsp70 loci.

A 280 kilobase (kb) contig was isolated from mouse genomic P1 and cosmid libraries, using as probes human cDNA and genomic DNA fragments that map in the interval between the second component of complement and tumor necrosis factor genes of the HLA complex. The clone contig demonstrates synteny of eleven mouse genes that are homologous to genes initially mapped within the human major histocompatibility complex. These include the mouse homologs of BAT2 (HLA-B-associated transcript 2) through BAT9 and also three HSP70-related genes. Five P1 clones form a contig of 240 kb that spans from BAT9 through BAT3. Twelve cosmid clones are arranged in three contigs that confirm most of the structure of the P1 contig and link the mouse BAT3 homolog to the BAT2 homolog approximately 15 kb farther telomeric. Polymorphic DNA markers within the cloned region were used to map the cleft palate susceptibility-1 (Cps-1) locus to the interval between Hsp70.1 and BAT6 (valyl-tRNA synthetase). This refines the location of the Cps-1 locus to a 45 kb region contained in the H2-124 P1 insert.

Chromosomal localization of five murine HSP70 gene family members: Hsp70-1, Hsp70-2, Hsp70-3, Hsc70t, and Grp78.

The 70,000-D heat shock protein (HSP70) gene family includes both heat-inducible and constitutively expressed genes. We have mapped five murine HSP70 genes to specific sites on three separate chromosomes. Southern blot analysis of Chinese hamster x mouse somatic cell DNAs was used to assign the gene for the 78,000-D glucose-regulated protein (Grp78) to Chromosome (Chr) 2, the male germ cell-specific Hsp70-2 and Hsc70t genes to Chr 12 and Chr 17, respectively, and the heat-inducible Hsp70-3 gene also to Chr 17. Southern blot analysis of DNA from the progeny of two multilocus crosses confirmed the Grp78 location on Chr 2 and suggested the order: centromere-Vim-Abl-Grp78-Hc. Similar analysis also confirmed the initial Hsp70-2 assignment to Chr 12 with the order: Hsp70-2-Aat-Igh. The Hsp70-3 and Hsc70t genes on Chr 17, along with the heat-inducible Hsp70-1 gene, were further localized by Southern blot analysis of genomic clones to the H-2 histocompatibility region with the order: Hsp70-1-Hsp70-3-Hsc70t-Bat-6 (human G7a, valyl-tRNA synthetase).

A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.

Bacteriophage P1 genes involved in the recognition and cleavage of the phage packaging site (pac).

The packaging of bacteriophage P1 DNA is initiated by cleavage of the viral DNA at a specific site, designated pac. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type P1 DNA and DNA derived from various P1 amber mutants that are deficient in pac cleavage, two distinct genes, referred to as pacA and pacB, were identified. These genes appear to be coordinately transcribed with an upstream P1 gene that encodes a regulator of late P1 gene expression (gene 10). pacA is located upstream from pacB and contains the 161 base-pair pac cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa, respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the P1 packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pac cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the P1 packaging site. PacB extracts also contain a second P1 protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pac cleavage is yet to be determined.

Frameshift mutagenesis in bacteriophage T7.

We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3). We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene. These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene. We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype. This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis. These assays for frameshift mutagenesis performed in E. coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.

A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization.

Using the bacteriophage P1 cloning system, we have constructed a two to three times coverage, high-molecular-weight (HMW) genomic library from mouse C127 fibroblast cells. The library consists of about 127,500 clones with an average insert size of about 70 kb that are organized into 300 primary pools containing approximately 425 clones per pool. For screening purposes the primary pools are combined into secondary pools (approximately 4250 clones each) and tertiary pools (approximately 21,250 clones each). Screening is performed by the polymerase chain reaction (PCR) with DNA isolated from the secondary and tertiary pools. We have screened the library for 13 different mouse sequences and have detected 11. Clones generated from two of the eleven positive screens were isolated from the library (those containing the c-fos and G alpha i2 genes) and were further characterized. Direct double-stranded sequencing of DNA from P1 clones with primers bordering the insert provided sequence information from each end of the cloned DNA.

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.

Deletion mutagenesis independent of recombination in bacteriophage T7.

Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined. The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats. Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase. The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication. The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp. To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene. Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way. Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.

Genetic deletions between directly repeated sequences in bacteriophage T7.

DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10(-10), while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10(-6). The deletion events were independent of a host recA mutation.

A single-base change in gene 10 of bacteriophage T7 permits growth on Shigella sonnei.

Bacteriophage T7 can extend its host range to include Shigella sonnei D2 371-48 by a mutation called ss found in the T7 major capsid protein, the gene 10 product. We show that a single A-to-C transversion at position 23150 in the T7 genome is responsible for the T7 ss mutant phenotype that allows the phage to avoid DNA degradation and undergo productive infection. The ss mutation causes an amino acid substitution of proline for glutamine at position 61 of the 344-amino-acid T7 major capsid protein.

Persistent hypophosphatemia following parathyroidectomy in end-stage renal disease: report of three patients.

Immediate and persistent hypophosphatemia following subtotal parathyroidectomy, despite discontinuation of phosphate binders, developed in three chronic hemodialysis patients. Although the serum phosphorous level is regularly reduced by parathyroidectomy in such patients, prolonged hypophosphatemia has not previously been reported. This observation supports the concept that parathyroid overactivity in end-stage renal disease is a major determinant of hyperphosphatemia.