Oogenesis in the Apogamous Fern Pteris cretica

The events that characterize egg formation and maturation inPteris cretica were investigated using transmission electronmicroscopy and electron microscope microprobe analysis. Theydid not differ significantly from those described for sexuallyreproducing ferns. The significance of these findings is discussedin relation to current theories concerning phase change in ferns. Pteris cretica, fern, apogamy, agamospory, transmission electron microscopy, oogenesis     

Mapping the genomic location of the gene encoding alpha-amanitin resistance in vaccinia virus mutants.

To facilitate the determination of the genomic location of the vaccinia virus gene(s) encoding alpha-amanitin resistance (alpha r) (Villarreal et al., J. Virol. 51:359-366, 1984), a collection of alpha r, temperature-sensitive (ts) mutants were isolated. The premise of these experiments was that mutants might be found whose dual phenotypes were the result of a single or two closely linked mutations. Genetic analyses of the alpha rts mutant library revealed two mutants, alpha rts7 and alpha rts12, that apparently fit this criterion; in alpha rts7 the two lesions were indistinguishable, whereas in alpha rts12 the two mutations were closely linked but separable. Cloned vaccinia virus HindIII DNA fragments were used to marker rescue the temperature-sensitive phenotype of these two dual mutants. The temperature-sensitive lesion of alpha rts7 was rescued by the HindIII N fragment (1.5 kilobases), whereas alpha rts12 was rescued by the neighboring HindIII M fragment (2.0 kilobases). The progeny virions of the alpha rts7 HindIII-N rescue reverted to an alpha-amanitin-sensitive phenotype, whereas the alpha rts12 HindIII-M progeny were still resistant to the drug. Taken together, these data indicate that the gene encoding alpha-amanitin resistance maps to the HindIII N fragment and provides evidence for the existence of essential vaccinia virus genes in a region of the genome previously believed to be nonessential for replication in tissue culture. Biochemical analyses revealed that both mutants were capable of synthesizing DNA as well as early and late viral proteins at the permissive and nonpermissive temperatures. At the nonpermissive temperature alpha rts12 and alpha rts7 were unable to process the major core precursors P94 and P65 into VP62 and VP60.     

Developmental expression of rat transforming growth factor-alpha mRNA.

Expression of the gene encoding transforming growth factor-alpha (TGF alpha) was examined in developing rat embryos by using a cloned TGF alpha cDNA as a hybridization probe. Northern blot analysis of RNA isolated from whole fetuses revealed that TGF alpha mRNA was present at relatively high levels in 8- through 10-day-old embryos and then declined to the low or undetectable level, which is characteristic of adult tissues before birth. The level of TGF alpha mRNA present during early gestation was similar to that present in retrovirus-transformed cells in culture, suggesting that TGF alpha expression is not highly localized in the embryo. These observations are consistent with the hypothesis that TGF alpha plays a role in development, possibly as a fetal growth factor.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.     

Genotype and phenotype associations with drought tolerance in barley tested in North Africa

A review is presented of genetic strategies deployed in a 3-yr project on drought tolerance in barley. Data were collected on genetic, physiological and agronomic traits in non-irrigated and irrigated field trials in Egypt, Morocco and Tunisia. A wide range of barley germplasm (developed from African and European cultivars, adapted landraces and wild barleys) was tested, and positive traits were found in each gene pool. The contrasting environments of the three North African countries had major effects on plant/genotype performance. Genetic effects were also detected, as were genotype × environment interactions. A range of strategies were deployed to investigate the physiology and genetics of quantitative traits associated with field performance. Quantitative trait locus (QTL) analysis was performed using backcross lines, recombinant inbred lines and doubled haploid mapping populations. A detailed genetic map was generated in the Tadmor × (ER/Apm) recombinant inbred lines, an important mapping population specifically developed by ICARDA (Centre for Agricultural Research in Dry Areas) and CIMMYT (International Maize and Wheat Improvement Center) to study drought. Quantitative trait loci (QTLs) for grain yield and other important morphological and physiological traits were also identified in a population of doubled haploids derived from F2BCj plants from a cross between a cultivar and a wild barley accession. Significantly, the wild parental line was found to contribute a number of positive alleles for yield. Effects of major developmental genes could explain many of the responses observed. QTLs were found to cluster around major genes controlling flowering time (sghI), plant stature (sdwI and arie.GP) and ear type (vrsl), and it is highly likely that the associations represent pleiotropic effects. Some QTLs were associated with candidate genes such as dehydrins and rubisco activase. One of the most significant results was the identification and generation of material that out performed the best local standards in the three participating North African countries; the selected lines have now entered local breeding programmes. The strategies adopted provided information on physiological traits, genotypes and genetic markers that could be used for marker-assisted selection. Target QTLs and their associated genetic markers may be deployed in marker assisted selection programmes to match crop phenology to the field environment.