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L B Schook

Publications and source records attributed to L B Schook.

At least 37 records · Page 2Linked to original sources
PubMed1998

Livestock variation of linked microsatellite markers in diverse swine breeds.

A panel of nine framework microsatellites (MS) linked to the Calcium Release Channel (CRC) locus on swine chromosome 6 (SSC6) was developed from the consensus genetic map. MS were screened across groups of unrelated animals from Yorkshire, Hampshire, Duroc, Landrace and Meishan swine breeds. Unique MS alleles for Yorkshire, Duroc, Landrace and Meishan breeds, and statistically significant (P < .05) associations between breeds and allele frequencies were found for each MS. Although breed marker heterozygosities ranged from 0.0 (S0035 in Duroc) to 0.92 (S0087 in Meishan), Correspondence Analysis identified MS alleles uniquely associated with either the Meishan breed, western breeds or alleles common to all breeds. Furthermore, an overall marker heterozygosity of < 0.70 demonstrates the need for multiple MS panels to accommodate reduced within-breed differences for identification of quantitative trait loci (QTL), marker assisted selection (MAS) programs or parental identification in commercial breeds.

Alleles↗
PubMed1998

Designs of reference families for the construction of genetic linkage maps.

The reference family panel is the foundation of a gene mapping program because it affects the cost and quality of the genetic linkage maps, and should be designed to yield reliable linkage detection and locus ordering at minimal gene mapping cost. A map cost function was defined as the number of genotypes required per marker per unit of genome coverage and was used to obtain optimal designs with respect to linkage detection. An ordering reliability function was defined as the likelihood ratio of the most likely order to the second most likely order of genetic markers and was used to find optimal designs with respect to locus ordering. Optimum levels of recombination frequency were found to be in the neighborhood of 0.11-0.15 for linkage detection and were in the region of 0.05-0.20 for locus ordering. Therefore, recombination frequencies optimal for linkage detection are also optimal for locus ordering. Based on the optimal detection levels, sample size (number of offspring) and map cost requirements were derived for six representative designs, assuming gender-specific linkage maps and two alleles with equal frequency for each marker. The sample size required for linkage detection ranged from 168 to 432 offspring for full-sib designs and ranged from 350 to 600 offspring for half-sib designs depending on the family size and the target LOD score, with corresponding minimal map costs of 10-20 genotypes per marker per centiMorgan map coverage. Locus ordering generally requires more genotypes than linkage detection. For full-sib designs, meioses from both genders should be used for locus ordering even when the maps are gender-specific. For half-sib designs, additional families may be needed for locus ordering. Sample size for ordering closely linked loci as required by positional cloning were provided. Effects of family size, grandparents, and marker polymorphism on design efficiency were analyzed.

Chromosome Mapping↗
PubMed1998

Cytokine and cytotoxic pathways of NK cell rejection of class I-deficient bone marrow grafts: influence of mouse colony environment.

Mouse NK cells may use both cytokine, e.g. IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-12, and cytotoxic, e.g. perforin and Fas-FasL, pathways to reject incompatible bone marrow cell (BMC) grafts. To begin a dissection of these two major pathways, mice bearing deletional mutations of IFN-gamma, TNF-RI/II or perforin, or mice treated with mAb to IL-12, IFN-gamma or NK1.1 were irradiated and challenged with class I-deficient BMC grafts, a system in which only NK cells are the effector cells. Proliferation of the donor-derived cells was judged in terms of splenic incorporation of [125I]iododeoxyuridine 5 or 7 days after cell transfer. All of these mice maintained in a specific pathogen-free (s.p.f.) environment were able to reject the BMC, except those treated with anti-NK1.1 mAb. However, perforin deficient mice maintained in a conventional breeding facility failed to reject class I (Tap-1)-deficient marrow cells. Transfer of mice from the pathogen-free to the conventional facility resulted in a slow and incomplete loss of the ability to reject marrow cells. Thus, the breeding colony environment can elicit otherwise undetectable defects in the rejection ability of perforin-deficient NK cells. This report will hopefully alert those investigators who have only studied immune gene knockout mice in s.p.f. facilities and found no significant abnormalities.

Animals↗
PubMed1998

Resolving the genetics of resistance to infectious diseases.

The genetics of resistance to disease is an area of great interest in agriculturally important plant and animal species. Selective breeding for resistance to pathogens in plants, animals and insects has demonstrated that resistance and susceptibility to pathogens are controlled by both genetic and environmental factors. The immune loci causally involved in susceptibility and resistance to disease are currently unknown. However, novel enabling molecular technologies promise to assist in unravelling the genetics of the host response to infectious diseases in new ways, and ultimately to improve seed stock genetics.

Animals↗
PubMed1997

Mapping of microsatellite markers developed from a flow-sorted swine chromosome 6 library.

Swine Chromosome (Chr) 6-enriched libraries, generated with size-fractionated DNA isolated from chromosomes sorted by flow cytometry, have been used to develop new Chr 6 microsatellite markers. Chromosome isolation procedures were established to reproducibly prepare high quality chromosomes from phytohemagglutinin (PHA)-stimulated swine peripheral blood lymphocytes and to sort individual chromosomes after staining with Hoechst 33258 and chromomycin A3. Chromosome purity was verified by specific staining of swine Chr 6 with fluorescence in situ hybridization (FISH) by use of painting probes generated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of as few as 300 sorted Chr 6. For library construction, DNA was extracted from flow-sorted pools representing Chr 6, amplified, size selected for fragments from 300 to 700 bp, and ligated into pBluescript SK II+ or Lambda ZAP Express. The libraries were then screened with a radiolabeled poly-(dCA) DNA probe. Of 107 (CA)n repeat-containing clones verified by sequencing, 21 were polymorphic and used to genotype the University of Illinois swine reference families. Linkage analysis was then performed with CRIMAP 2.4 (LOD > 3.0), and the results showed that 15 of the microsatellites mapped to swine Chr 6. At least three of these new markers map to locations where there were gaps in the consensus Chr 6 map. Another four markers, because of their PIC values, should provide more informative markers in other areas of the map. Most of the new markers can also be used for automated genotyping with fluorescent labeling. This set of 15 new Chr 6 markers will, therefore, be useful in helping to define QTL associated with swine Chr 6.

Animals↗
PubMed1997

Development and use of a microdissected swine chromosome 6 DNA library.

To facilitate the identification of microsatellite genetic markers from a single swine chromosome, chromosome microisolation and microcloning have been used to generate a swine chromosome 6-specific DNA library. Ten copies of swine chromosome 6 were scraped from metaphase spreads, ligated to custom-prepared adaptors, and amplified by PCR. The purity of the amplified product was verified by fluorescent in situ hybridization. The utility of the chromosome painting probe for heterologous painting was demonstrated and confirmed that swine chromosome 6 is syntenic to human chromosomes 1p and 19q. A small insert genomic library of 1.39 x 10(6) clones was generated from the PCR-amplified chromosome 6 genomic DNA and screened for (GT)n microsatellite genetic markers. Nine (GT)n microsatellite markers were developed and genotyped on a Yorkshire x Meishan swine reference family. All nine markers genetically mapped to chromosome 6, confirming the purity of the microisolation method. The method used here should be adaptable to the microdissection of subchromosomal regions of not only the swine genome but also other livestock genomes.

Animals↗
PubMed1995

Differential expression of novel genes by bone marrow-derived macrophage populations.

In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-gamma in GM-CSF-derived bone marrow macrophage (m phi). M theta were treated with 50 U/ml IFN-gamma for 40, 70 and 140 min to induce expression of early genes regulated by IFN-gamma, and the M phi were pooled. Poly(A)+RNA was prepared from both unactivated and IFN-gamma-stimulated m theta, and cDNA libraries were constructed in lambda ZAP. Genes expressed in common by both m theta populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated m phi as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-gamma-activated GM-CSF- and CSF-1-derived m phi. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-gamma treatment of GM-CSF-derived m phi, but demonstrated high constitutive transcript levels in CSF-l-derived m phi. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived m phi, but not by CSF-1-derived m phi, even after activation by IFN-gamma. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived m phi resides in the differential expression of early genes specifically induced by IFN-gamma.

Animals↗
PubMed1995

Directed integration of the physical and genetic linkage maps of swine chromosome 7 reveals that the SLA spans the centromere.

The first integrated physical and genetic linkage map encompassing the entire swine chromosome 7 (SSC7) reveals that the porcine MHC (SLA) spans the centromere. A SLA class II antigen gene lies on the q arm, whereas class I and III genes lie on the p arm, suggesting that the presence of a centromere within the SLA does not preclude a functional complex. The SLA appears smaller than other mammalian MHC, as the genetic distance across two class I, three class II, and three class III SLA gene markers is only 1.1 cM. There are significant variations in recombination rates as a function of position along the chromosome, and the SLA lies in the region with the lowest rate. Furthermore, the directed integration approach used in this study was more efficient than previous efforts that emphasized the screening of large insert libraries for random microsatellites.

Animals↗
PubMed1995

Polymerase chain reaction based genotyping for characterization of SLA-DQB and SLA-DRB alleles in domestic pigs.

Molecular genotyping of swine major histocompatibility complex SLA-DQB and SLA-DRB genes using polymerase chain reaction (PCR)-based amplification is described. Locus-specific oligonucleotide primers were designed for the analysis of expressed SLA genes by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products were sequenced, and the information gained was used to design primers for PCR genotyping of the exon 2 (beta 1) region from genomic DNA templates. A single segregating amplification product was detected for both DQB and DRB in all animals. PCR products were digested with restriction enzymes. Seven SLA-DQB PCR-restriction fragment length polymorphism (RFLP) pattern types were observed for both HaeIII and RsaI that defined 14 SLA-DQB alleles. A total of seven SLA-DRB PCR-RFLP pattern types were defined using MspI (3 RFLP pattern types) and RsaI (6 RFLP pattern types). In order to demonstrate their universal utility, the primers were tested on genomic DNA samples from 10 different swine breeds. No breed-specific alleles were observed. These results show that locus-specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA-DQB and SLA-DRB from genomic DNA.

Alleles↗
PubMed1995

First international workshop on porcine chromosome 6. Report and abstracts.

Recent advances in the use of microsatellite markers and the development of comparative gene mapping techniques have made the construction of high resolution genetic maps of livestock species possible. Framework and comprehensive genetic linkage maps of porcine chromosome 6 have resulted from the first international effort to integrate genetic maps from multiple laboratories. Eleven highly polymorphic genetic markers were exchanged and mapped by four independent laboratories on a total of 583 animals derived from four reference populations. The chromosome 6 framework map consists of 10 markers ordered with high local support. The average marker interval of the framework map is 15.1 cM (sex averaged). The framework map is 135, 175 and 109 cM in length (for sex averaged, female and male maps, respectively). The comprehensive map includes a total of 48 type I and type II markers with a sex averaged interval of 3.5 cM and is 166, 196 and 126 cM (for sex averaged, female and male maps, respectively). Additional markers within framework map marker intervals can thus be selected from the comprehensive map for further analysis of quantitive trait loci (QTL) located on chromosome 6. The resulting maps of swine chromosome 6 provide a valuable tool for analysing and locating QTL.

Animals↗
PubMed1995

Nuclear migration of NF-kappa B correlates with TNF-alpha mRNA accumulation.

Previous work on the transcriptional regulation of the mouse TNF-alpha promoter had indicated a major role for the NF-kappa B transcription factor in the induction of the gene by endotoxin. However, similar studies using the human promoter failed to establish a role for this factor. We measured the nuclear migration of NF-kappa B and the accumulation of TNF-alpha mRNA in murine T lymphoblasts and bone marrow-derived macrophages (BMDM) under different activation conditions, seeking to establish a correlation. Activation of NF-kappa B and accumulation of TNF-alpha mRNA correlated semiquantitatively under the following conditions: (1) inhibition of NF-kappa B by dithiocarbamates; (2) induction of TNF synthesis by taxol; (3) partial induction of TNF-alpha mRNA by various inducers in macrophages from lpsd mice; and (4) inhibition of NF-kappa B activation by a protease inhibitor. However, inhibition of TNF-alpha mRNA accumulation by cAMP inducers had no effect on NF-kappa B induction. We conclude that NF-kappa B translocation is necessary, but not sufficient for the transcriptional induction of the TNF-alpha gene by LPS in macrophages or by phorbol ester and ionomycin in T lymphocytes.

Animals↗
PubMed1995

Endotoxin and interferon-gamma differentially regulate the transcriptional levels of proto-oncogenes and cytokine genes during the differentiation of colony-stimulating factor type-1-derived macrophages.

The expression of cytokine genes for interleukin-1 (IL-1) (alpha and beta) and tumour necrosis factor-alpha (TNF-alpha), along with the proto-oncogenes c-fos, c-fms and c-myc, was examined by nuclear run-off and Northern blot analysis during in vitro differentiation of colony-stimulating factor type-1 (CSF-1)-derived bone marrow macrophages (BMDM). Constitutive transcription of c-myc was maximal on day 3 and decreased with differentiation. Constitutive transcription of c-fms and c-fos was similar at all times examined. In contrast, the steady-state mRNA levels were maximal on day 5 for c-myc and day 7 for c-fms and c-fos. Thirty minutes after endotoxin (lipopolysaccharide; LPS) stimulation, there was a rapid increase in run-off transcription rates for c-myc in day 3-day 9 BMDM, with maximal levels observed in day 7 BMDM. c-fms transcription was maximally induced within 1 hr by LPS in day 3 and day 5 BMDM. LPS induced transcription of c-fos to equivalent levels in day 3-day 9 BMDM. LPS stimulation augmented steady-state mRNA levels for c-myc, c-fms and c-fos. Maximal induction of c-myc was observed in day 3 BMDM. c-fos and c-fms were both maximally induced in day 5 and day 7 BMDM. Interferon-gamma (IFN-gamma) had no effect on transcription of the proto-oncogenes examined. In contrast to the proto-oncogenes, peak levels of run-off transcription for IL-1 alpha and IL-1 beta genes were observed 1-2 hr after LPS stimulation for day 3, day 5 and day 7 BMDM. The kinetics of LPS-induced steady-state mRNA accumulation of IL-1 alpha and IL-1 beta were similar to the kinetics of run-off transcription. Constitutive transcription of TNF-alpha was observed on all days of differentiation. LPS and IFN-gamma both enhanced run-off transcription of the TNF-alpha gene; however, LPS had a more pronounced effect. The kinetics of induction of TNF-alpha transcription paralleled the kinetics of steady-state TNF-alpha mRNA accumulation. IFN-gamma resulted in secretion of TNF-alpha in day 5, day 7, and day 9 BMDM after 4-8 hr of stimulation. Day 3 BMDM had little, if any, secreted TNF-alpha activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Specific transcriptional inhibition of bone marrow-derived macrophage tumor necrosis factor-alpha gene expression and protein production using novel enantiomeric carbocyclic nucleoside analogues.

Tumor necrosis factor-alpha (TNF-alpha) is a powerful macrophage-derived proinflammatory cytokine, via both direct effects on host tissues as well as indirectly through the induction of other proinflammatory mediators, including interleukin- (IL) 1 beta and IL-6. Activation of murine bone marrow-derived macrophages (BMDM phi) with lipopolysaccharide (LPS) causes rapid expression of TNF-alpha, which as an autocrine factor enhances BMDM phi function through IL-1 beta and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDM phi TNF-alpha using novel enantiomeric carbocyclic nucleoside analogues. BMDM phi were derived in vitro from murine bone marrow progenitors using colony stimulating factor-1 and treated with combinations of LPS (1-100 nG/ml) and the enantiomeric carbocyclic nucleoside (10-100 microM) analogues MDL 201, 112 (9-[(1S,3R)-cis-cyclopentan-3-ol]adenine); MDL 201,451 (9-[1R,3S)-cis-cyclopentan-3-ol]adenine); MDL 201,449 (9-[(1R,3R)-trans-cyclopentan-3-ol]adenine) and MDL 201,484 (9-[(1S,3S)-trans-cyclopentan-3-ol]adenine). Northern blot analysis showed that MDL 201,449 was the most effective agent in vitro at selectively inhibiting TNF-alpha. MDL 201,449 reduced TNF-alpha mRNA levels by nearly 50% for up to 4 hr after the simultaneous addition of LPS and the synthetic agent. In contrast, mRNA and secreted protein levels for IL-1 beta (measured by the D10.S bioassay) and mRNA for TNF-alpha receptor p60 and TNF-alpha receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDM phi up-to 2 hr after LPS treatment and at concentrations as low as 10 microM. Regulation of BMDM phi IL-6 by carbocyclic nucleoside analogues in response to LPS appears to be both concentration and time dependent, because IL-6 mRNA and secreted protein levels were inhibited at only high drug concentrations (100 microM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that M phi-derived proinflammatory cytokine gene expression is differentially, rather than coordinately, regulated by selective signal transduction and/or molecular pathways. Enantiomeric carbocyclic nucleoside analogues that specifically inhibit TNF-alpha may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-alpha has been shown to have an important role in initiating the early stages of disease.

Adenine↗
PubMed1994

Cytokine regulation of TNF-alpha mRNA and protein production by unprimed macrophages from C57Bl/6 and NZW mice.

It is well known that bacterial lipopolysaccharide (LPS) induces the synthesis of tumor necrosis factor alpha (TNF-alpha) and other inflammatory cytokines by primary monocytes and macrophages and that the Th1 lymphokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), augment this response. We investigated the ability of IL-2 and IFN-gamma to induce the production of TNF-alpha mRNA and protein independently of LPS and the modulation of this response by macrophage colony-stimulating factor (M-CSF) and IL-10. We found that IL-2 and IFN-gamma were both able to induce the accumulation of TNF-alpha mRNA, albeit with slower kinetics than LPS, and that they acted synergistically. However, very little TNF bioactivity was secreted by lymphokine-stimulated macrophages unless LPS was also added. This finding underscores the importance of translational effects in the control of TNF production. M-CSF and IL-10 strongly inhibited TNF production at the level of both mRNA and bioactivity but had no effect on the production of IL-6. Bone marrow-derived or thioglycollate-elicited macrophages from the NZW mouse strain, which have been reported to be deficient in their ability to produce TNF, were at least as responsive to LPS or lymphokines as those taken from the C57Bl/6 strain and were similarly affected by M-CSF and IL-10. Therefore, the genetic defect of NZW mice is not a primary deficiency in TNF production.

Animals↗