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P E Klein

Publications and source records attributed to P E Klein.

9 recordsLinked to original sources
PubMed2002

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

Chromosome Mapping↗
PubMed2001

Mapping genes on an integrated sorghum genetic and physical map using cDNA selection technology.

Sorghum is an important target of plant genomics. This cereal has unusual tolerance to adverse environments, a small genome (750 Mbp) relative to most other grasses, a diverse germplasm, and utility for comparative genomics with rice, maize and other grasses. In this study, a modified cDNA selection protocol was developed to aid the discovery and mapping of genes across an integrated genetic and physical map of the sorghum genome. BAC DNA from the sorghum genome map was isolated and covalently bound in arrayed tubes for efficient liquid handling. Amplifiable cDNA sequence tags were isolated by hybridization to individual sorghum BACs, cloned and sequenced. Analysis of a fully sequenced sorghum BAC indicated that about 80% of known or predicted genes were detected in the sequence tags, including multiple tags from different regions of individual genes. Data from cDNA selection using the fully sequenced BAC indicate that the occurrence of mislocated cDNA tags is very low. Analysis of 35 BACs (5.25 Mb) from sorghum linkage group B revealed (and therefore mapped) two sorghum genes and 58 sorghum ESTs. Additionally, 31 cDNA tags that had significant homologies to genes from other species were also isolated. The modified cDNA selection procedure described here will be useful for genome-wide gene discovery and EST mapping in sorghum, and for comparative genomics of sorghum, rice, maize and other grasses.

Base Sequence↗
PubMed2000

A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map.

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones ( approximately 4x genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified approximately 2400 BACs and ordered approximately 700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located approximately 200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in approximately 65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.

Chromosomes, Bacterial↗
PubMed1998

Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit nepsilon-methyltransferase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.

Amino Acid Sequence↗
PubMed1986

Reduction of pupillary constriction during cataract surgery using suprofen.

The efficacy of a new nonsteroidal anti-inflammatory agent, suprofen, for reducing pupillary constriction during cataract surgery was ascertained in a double-masked, multicenter, clinical study. Prior to surgery 1.0% suprofen or a placebo was instilled; the surgeon's normal regimen of mydriatics and cycloplegics was used. Suprofen (209 patients) was far more effective than the placebo (203 patients) in maintaining a dilated pupil prior to intraocular lens (IOL) implantation (or instillation of a miotic). The mean pupillary area prior to IOL implantation was 6.3 sq mm larger (20% larger) in patients treated with suprofen than in patients receiving the placebo. The investigators' subjective evaluations of the adequacy of pupil size for IOL implantation and of the difficulty of IOL implantation favored patients treated with suprofen over those receiving the placebo.

Adult↗
PubMed1986

Clinical and immunological results of corneal allograft rejection.

Of 111 episodes of graft rejection in 66 patients, 62 responded to therapy with graft clearing (responders); 49 did not (non-responders). Both groups were of similar age, sex, and etiology; both had a similar rate of glaucoma and a similar rate of previous grafting. In responders the graft reaction was shorter in duration (2.2 vs. 5.6 wks. p less than 0.005), and it was necessary to increase the number of glaucoma medications more often in non-responders compared to responders (41% vs. 19%, p less than 0.02). The interval from surgery to reaction was similar in responders and non-responders (18.2 vs. 13.3 mos., p greater than 0.1). An epithelial rejection line was present in 11% of responders, but was not present in non-responders (p less than 0.05). Lymphocytotoxic antibody development correlated with rejection in 16 of 64 episodes. Patients who responded to treatment were more frequently asymptomatic (p less than 0.05) or were treated earlier following the onset of symptoms compared to non-responders (p less than 0.0001).

Adult↗
PubMed1982

Posttreatment drinking behavior among inpatients from an industrial alcoholism program.

Findings from a follow-up study of inpatients from an industrial alcoholism program are presented. At 3-month follow-up, 46% were abstinent and 22% drinking moderately; at 9 months, 37% were abstinent and 20% drinking moderately. Moderate drinkers at 3 months had a high relapse rate by 9 months. In comparing moderate drinkers at 9 months to abstainers and to those drinking destructively, it was found that moderate drinkers were more similar to abstainers than to destructive drinkers on most variables. Relationships between drinking outcomes and several sets of predictor variables are presented and discussed. From these data it is evident that social support (being married, family involved in treatment, AA and/or religious involvement) is crucial in recovery from alcoholism, that employer's involvement had a positive influence, that coercion for treatment did not have a negative impact, that prior job problems had a delayed negative impact, and that treatment outcome for earlier-phase alcoholics was relatively poor in comparison to "chronic" cases in this program.

Alcohol Drinking↗
PubMedDate not supplied

A high-density genetic map of Sorghum bicolor (L.) Moench based on 2926 AFLP, RFLP and SSR markers.

Using AFLP technology and a recombinant inbred line population derived from the sorghum cross of BTx623 x IS3620C, a high-density genetic map of the sorghum genome was constructed. The 1713 cM map encompassed 2926 loci distributed on ten linkage groups; 2454 of those loci are AFLP products generated from either the EcoRI/MseI or PstI/MseI enzyme combinations. Among the non-AFLP markers, 136 are SSRs previously mapped in sorghum, and 203 are cDNA and genomic clones from rice, barley, oat, and maize. This latter group of markers has been mapped in various grass species and, as such, can serve as reference markers in comparative mapping. Of the nearly 3000 markers mapped, 692 comprised a LOD >3.0 framework map on which the remaining markers were placed with lower resolution (LOD <3.0). By comparing the map positions of the common grass markers in all sorghum maps reported to date, it was determined that these reference markers were essentially collinear in all published maps. Some clustering of the EcoRI/MseI AFLP markers was observed, possibly in centromeric regions. In general, however, the AFLP markers filled most of the gaps left by the RFLP/SSR markers demonstrating that AFLP technology is effective in providing excellent genome coverage. A web site, http://SorghumGenome.tamu.edu, has been created to provide all the necessary information to facilitate the use of this map and the 2590 PCR-based markers. Finally, we discuss how the information contained in this map is being integrated into a sorghum physical map for map-based gene isolation, comparative genome analysis, and as a source of sequence-ready clones for genome sequencing projects.

Chromosome Mapping↗