Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma.

Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.     

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter--glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to wound signals in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.     

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximatly tenfold in the presence of oxygen and three- to fourfold under heatshock conditions.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.     

Use of seedling progeny tests for genetical studies as part of a potato (Solanum tuberosum subsp. tuberosum) breeding programme

A diallel set of crosses, including selfs and some reciprocal crosses, was made between 15 parents chosen for their male fertility from those included in a tetraploid potato (Solanum tuberosum subsp. tuberosum) breeding programme at the Scottish Crop Research Institute. Seedling progeny tests were used to evaluate the progenies for non-race-specific resistance to late blight (Phytophthora infestans) in both foliage and tubers, quantitative resistance to the white potato cystnematode (PCN) (Globodera pallida) and the commercial worth of their tubers as judged by breeders' visual preference. No reciprocal differences were found. Comparisons of the selfs and crosses revealed inbreeding depression for breeders' preference, which varied among the parents from negligible to severe, whilst there were also statistically significant differences for foliage and tuber blight, but not for PCN. When the selfs were omitted from the combining ability analyses, large differences in general combining ability (GCA) were found for all four traits, and smaller differences in specific combining ability for tuber blight and breeders' preference. The only statistically significant correlation between GCAs for different traits was a favourable one of r = 0.56 between foliage and tuber resistance to late blight. It was concluded that prospects were good for simultaneously improving all four traits by multitrait genotypic recurrent selection.     

Evolutionary Divergence of the Genetic Architecture Underlying Photoperiodism in the Pitcher-Plant Mosquito, Wyeomyia Smithii

We determine the contribution of composite additive, dominance, and epistatic effects to the genetic divergence of photoperiodic response along latitudinal, altitudinal, and longitudinal gradients in the pitcher-plant mosquito, Wyeomyia smithii. Joint scaling tests of crosses between populations showed wide-spread epistasis as well as additive and dominance differences among populations. There were differences due to epistasis between an alpine population in North Carolina and populations in Florida, lowland North Carolina, and Maine. Longitudinal displacement resulted in differences due to epistasis between Florida and Alabama populations separated by 300 km but not between Maine and Wisconsin populations separated by 2000 km. Genetic differences between New Jersey and Ontario did not involve either dominance or epistasis and we estimated the minimum number of effective factors contributing to a difference in mean critical photoperiod of 5 SD between them as n(E) = 5. We propose that the genetic similarity of populations within a broad northern region is due to their more recent origin since recession of the Laurentide Ice Sheet and that the unique genetic architecture of each population is the result of both mutation and repeated migration-founder-flush episodes during the dispersal of W. smithii in North America. Our results suggest that differences in composite additive and dominance effects arise early in the genetic divergence of populations while differences due to epistasis accumulate after more prolonged isolation.