Heparin-binding angiogenesis factors: detection by immunological methods

Immunological methods for the detection of basic fibroblast growth (FGF) are described. Polyclonal antibodies directed against synthetic peptides representing amino-, internal, and carboxy-terminal regions of basic FGF, were raised in rabbits. Five techniques, enzyme-linked immunosorbent assay, immunoblot, electrophoretic transfer (Western) blot, immunoprecipitation and radioimmunoassay, were used to detect basic FGF. These techniques were used to demonstrate that a human hepatoma cell line synthesizes a growth factor structurally related to brain and pituitary basic FGF.     

Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F₁ progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and β-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F₁ generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F₁ progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Interval mapping of quantitative trait loci affecting NESTUR, a stem growth efficiency index of radiata pine seedlings

 NESTUR (needle-to-stem unit rate) is a stem growth index of conifer seedlings that measures the efficiency of stemwood production per unit of needle growth, and is related to other seedling traits such as height, stem diameter, stem volume and needle volume. Quantitative trait loci (QTLs) affecting the expression of stem growth efficiency in radiata pine seedlings were investigated using a RAPD linkage map constructed from markers scored on haploid, megagametophytic DNA. Four putative QTLs were detected which accounted for 8.5–36.4% of the population variance. A search for evidence of epistasis, using both complete pairwise and conditional interactions, did not yield any statistically significant result. Over a 3-year period, seedlings with high-NESTUR marker alleles showed a superior growth performance of 17–40% for height, diameter and volume over those with low-NESTUR marker alleles. Received: 10 July 1997 / Accepted: 31 March 1998     

Ischemic Preconditioning in the Liver Is Independent of Regulatory T Cell Activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.     

Comparative mapping in loblolly and radiata pine using RFLP and microsatellite markers

Genetic linkage maps were constructed for loblolly pine (Pinus taeda L.) and radiata pine (P. radiata D. Don) using a common set of RFLP and microsatellite markers. The map for loblolly pine combined data from two full-sib families and consisted of 20 linkage groups covering 1281 cM. The map for radiata pine had 14 linkage groups and covered 1223 cM. All of the RFLP probes readily hybridise between loblolly and radiata pine often producing similar hybridisation patterns. There were in total 60 homologous RFLP loci mapped in both species which could be used for comparative purposes. A set of 20 microsatellite markers derived from radiata pine were also assayed; however, only 9 amplified and revealed polymorphic loci in both species. Single-locus RFLP and microsatellite markers were used to match up linkage groups and compare order between species. Twelve syntenic groups were obtained each consisting of from 3 to 9 homologous loci. The order of homologous loci was colinear in most cases, suggesting no major chromosomal rearrangements in the evolution of these species. Comparative mapping between loblolly and radiata pine should facilitate genetic research in both species and provide a framework for mapping in other pine species. Received: 25 November 1998 / /Accepted: 19 December 1998     

Age-related changes in the expression of QTLs for growth in radiata pine seedlings

 Haploid megagametophytes from a full-sib cross of Pinus radiata were used to construct a genetic linkage map for radiata pine based on random amplified polymorphic DNA (RAPD) markers. The map, which was made from 222 markers, was used to carry out a QTL analysis of growth using measurements made on seedlings from which these megagametophytes were collected at an early germination stage. Trends in the expression of QTLs for stem diameter, volume and height were compared using measurements made at 5 months of age, and at 1, 2 and 3 years of age. None of the observed trends showed complete stability, i.e. none of the putative QTL positions detected at any one age was strongly expressed at all of the four stages of measurement. However, 45% of the trends showed partial stability, i.e. putative QTLs significant at one age were also detected at a subsequent age. Trends in QTL expression with age followed one of three patterns: (1) putative QTLs at some locations showed a gradual linear increase in influence from 5 months of age and were highest at 3 years of age; (2) QTLs detected at 5 months of age gradually became less significant with age; and (3) some putative QTLs showed a curvilinear increase in effect from 5 months of age, reaching their peak expression at 1 to 2 years, and sometimes were still detected at 3 years of age. Received: 10 July 1997 / Accepted: 31 March 1998     

Linkage of RAPD markers to NESTUR, a stem growth index in radiata pine seedlings

 Needle-to-stem unit rate (NESTUR) is a stem growth index of conifer seedlings that measures the efficiency of stemwood production per unit of foliage growth. The random amplified polymorphic DNA (RAPD) technique was applied to haploid DNA from the megagametophytes of a full-sib radiata pine cross to find markers linked to factors controlling the NESTUR trait. Using the bulked segregant analysis approach, 23 of 933 primers displayed putative linkage to factors controlling NESTUR. Based on the genotypic analysis of 174 individuals, two quantitative trait loci (QTLs) controlling NESTUR were identified at ANOVA P-levels of 0.01–0.001. The QTLs were identified by RAPD markers OPE-06₄₅₀ and OPA-10₁₂₀₀, which were linked to each other (r=7%), and UBC-333₅₅₀, which was not linked to the other two. Linkage to components of NESTUR (increments in stem diameter and stem volume) was demonstrated for UBC-333₅₅₀, while the others were not linked to NESTUR components. Received: 18 December 1996/Accepted: 24 January 1997     

Use of recombinant substitution lines in the construction of RFLP-based genetic maps of chromosomes 6A and 6B of tetraploid wheat (Triticum turgidum L.)

RFLP-based genetic maps of chromosomes 6A and 6B of Triticum turgidum have been constructed using data obtained by the study of Triticum turgidum var durum cv Langdon-T. t. var dicoccoides recombinant substitution lines (RSLs) supplemented with data obtained from F₃ families derived from Langdon dicoccoides 6A and 6B disomic substitution lines. The average RFLP frequencies detected for the two chromosomes in a test of 45 DNA clones with six restriction enzymes were 56% and 53%, respectively, and a subset of 32 clones gave frequencies of 75% and 72%, respectively. Seventeen loci were mapped in 6A and 18 in 6B. With the possible exception of 5 loci in the centromeric region of 6A, all of the mapped 6A and 6B loci are located in the same arm as are homologous loci in hexaploid wheat, and the linear order of the loci is the same in the two chromosomes, except possibly close to the centromere. Major differences in genetic distances exist between homologous loci located in the proximal regions of the 6AL and 6BL linkage groups, however, the distances being much larger in the former than in the latter. The 6B maps that were constructed using data from both the RSL and the F₂ populations and using data from the RSL population alone closely resemble one another, indicating that the 6B RSL population, composed of 85 lines, can be reliably used for genetic mapping. Additional studies must be conducted before the utility of the 6A RSL population, composed of 66 lines, can be adequately assessed.