Expression of murine adenosine deaminase (ADA) in transgenic maize.

A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta or the adenosine analogue 2'-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41-kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Culltures expressing ADA grew in the presence of 200 mg/l 2'-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progency of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.

Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambdaN+cI[Ts]cro- prophage in E. coli cells shifted from 30 degreesC to 42 degreesC, accompanied by loss of immlambda and Rex+ phenotypes and emergence of a non-immune exclusion-state.

The majority of bacteria, which carry the N+-cI857[Ts]-cro--O+-P+ fragment of lambda genome, are killed when derepressed by shifting from 30 degreesC to 42 degreesC. Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical immlambda-immunity phenotype when grown at 30 degreesC; however, when shifted from 30 degreesC to 42 degreesC, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype). We also found that the immlambda survivor cfu quickly lost their Rex+ exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degreesC to 42 degreesC, even though they produced CII, which stimulates pE-cI-rexA-rexB transcription. The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambdawt, and the heteroimmune phage, lambdaimm434. However, lambdavir and spontaneous mutants of lambdawt (lambdase mutations localized within oR) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degreesC. Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes. Phage like lambdawt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny. Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded. The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s). We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degreesC. In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment. The remaining survivor cfu had multiple base substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent. In some of these mutants, either an ochre nonsense mutation or a single-base frameshift deletion inactivated P.

Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis.

Schizosaccharomyces pombe cells divide by medial fission. One class of cell division mutants (cdc), the late septation mutants, defines four genes: cdc3, cdc4, cdc8, and cdc12 (Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Mol. & Gen. Genet. 146:167-178). We have cloned and characterized the cdc4 gene and show that the predicted gene product. Cdc4p, is a 141-amino acid polypeptide that is similar in sequence to EF-hand proteins including myosin light chains, calmodulin, and troponin C. Two temperature-sensitive lethal alleles, cdc4-8 and cdc4-31, accumulate multiple nuclei and multiple improper F-actin rings and septa but fail to complete cytokinesis. Deletion of cdc4 also results in a lethal terminal phenotype characterized by multinucleate, elongated cells that fail to complete cytokinesis. Sequence comparisons suggest that Cdc4p may be a member of a new class of EF-hand proteins. Cdc4p localizes to a ringlike structure in the medial region of cells undergoing cytokinesis. Thus, Cdc4p appears to be an essential component of the F-actin contractile ring. We find that Cdc4 protein forms a complex with a 200-kD protein which can be cross-linked to UTP, a property common to myosin heavy chains. Together these results suggest that Cdc4p may be a novel myosin light chain.

Transpogenes: the transposition-like integration of short sequence DNA into the yeast 2 micron plasmid creates the STB locus and plasmid-size polymorphism.

The type-2 2 mu plasmid of industrial yeast strains exhibits extensive size polymorphism in the STB (plasmid stability) locus and IR (inverted repeat)-right region. Comparative DNA sequence analyses of STB alleles identified a 38-bp sequence flanked by a 25-bp direct repeat as the underlying structural motif. Variable unequal recombination within the direct repeat accounted for the observed polymorphism of STB alleles. IR-right polymorphism was observed to result from tandem duplication of a 22-bp sequence flanked by a 9-bp direct repeat. The flanking direct repeats marked both loci as originating from the transposition-like integration of short DNA fragments. We call these structures transpogenes and note that these are hybrid structures of host and foreign DNA which can evolve into functional loci.

Chimeric evolution of the 2-microns genome in Saccharomyces cerevisiae.

We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 microns) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-microns variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-microns plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection.

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups.

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weakly-virulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.

Modified binary plant transformation vectors with the wild-type gene encoding NPTII.

The defective gene encoding neomycin phosphotransferase (NPTII) present in the binary plasmid vector, pBin19, was replaced with the wild-type (wt) gene. Plasmid vectors analogous to pBin19, pBI121 and pBI101 were constructed carrying the gene encoding the wt NPTII enzyme activity.

Characterization of an Escherichia coli gene encoding betaine aldehyde dehydrogenase (BADH): structural similarity to mammalian ALDHs and a plant BADH.

An open reading frame of 1476 nucleotides, cloned from a region of the Escherichia coli genome encoding betaine biosynthesis functions, was shown to encode a betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8). Either of two adjacent codons (5'-GTGATG) could function as a start codon, producing a presumptive polypeptide of 491 or 490 amino acids. The deduced primary structure of the E. coli BADH showed 39-43% positional identity, over its entire length, to aldehyde dehydrogenases (ALDH: EC 1.2.1.3) of mammalian origin. This similarity increased to 75-77% when conservative aa substitutions were also taken into consideration. Spinach BADH was also similar to the bacterial BADH, showing 38% identity and 80% overall similarity. Other homologs included a fungal and a putative bacterial ALDH. Although E. coli BADH was specific for the substrate, betaine aldehyde, it showed the highest levels of similarity to the prototype human ALDH-2. Only one gap in each sequence had to be introduced for optimal alignment. The conservation between E. coli BADH and the ALDHs was also evident in the predicted secondary structures and hydrophilicity profiles of the polypeptides, suggesting a similarity in the overall folding patterns of ALDH and BADH. These observations suggest a common ancestry for BADH and ALDH, preceding prokaryote-eukaryote divergence.

Sequence diversity of yeast 2 microns RAF gene and its co-evolution with STB and REP1.

Despite the extensive study of yeast 2 microns plasmid, the exact function of plasmid-encoded RAF gene is not clear. Variants of 2 microns plasmids from industrial Saccharomyces cerevisiae yeasts were isolated and characterized. Sequencing of RAF alleles revealed about 8% nucleotide and 10% amino acid diversities between 2 microns variants of closely related strains, RAF sequence variations were correlated with STB-REP1 sequence diversity. We also used restriction fragment length polymorphism linkage to screen a large number of yeast strains from different fermentation industries. The results clearly show a tight linkage of STB-REP1-RAF variations. Thus, our observations suggest that plasmid-borne cis- and trans-acting elements co-evolved to form an optimal molecular parasite and that RAF may play a role in active plasmid partitioning.

A bifunctional fusion between beta-glucuronidase and neomycin phosphotransferase: a broad-spectrum marker enzyme for plants.

We have used an in vivo selection approach to isolate a gene encoding a bifunctional fusion peptide between Escherichia coli beta-glucuronidase (GUS) and neomycin phosphotransferase II (NPT-II) from transposon Tn5 in the NH2-GUS::NPT-II-COOH configuration. The fused gene is predicted to encode a fusion peptide 885 amino acids long, and was shown in E. coli to synthesize a 97-kDa GUS+ NPT-II+ gene product. Gel-filtration chromatography suggested that, while the native GUS may be active as a dimer and NPT-II as a monomer, the elution profile of the fusion protein is consistent with that of a trimer. The fusion marker has been produced and defined in transgenic Nicotiana tabacum plants, where both the chimeric gene and the gene product were stable. The bifunctional gene enabled direct KmR selection at the callus stage and enzymatic or histochemical assessment of the steady-state production of GUS activity in regenerated plants. In addition to allowing structure-function determination for the GUS and NPT-II domains of the fusion peptide, the gus::npt-II gene simplifies vector constructs where both marker domains are desired.

DNA sequence divergence and functional conservation at the STB locus of yeast 2 microns circle variants.

2 microns DNA isolated from industrial Saccharomyces cerevisiae yeasts exhibited extensive restriction fragment length polymorphisms. At least five 2 microns species were identified from eleven [cir+] strains. Southern hybridization mapped restriction fragment length polymorphisms at STB, a cis-acting locus essential for plasmid partitioning. Some 2 microns variants (e.g., 4110-2 microns and 4108-2 microns) had an altered number of 125-bp consensus repeats at STB. However, the corresponding region of 7754-2 microns has only approximately 70% nucleotide sequence homology with the 125-bp STB consensus repeat. YRp plasmids containing 7754-2 microns STB behave as YEp plasmids in laboratory yeasts, thereby indicating STB sequence divergence coupled to conservation of function.

Evidence for cis- and trans-acting element coevolution of the 2-microns circle genome in Saccharomyces cerevisiae.

We compared the DNA sequence of the yeast 2-microns plasmid cis-acting STB and transacting REP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a unique STB sequence (type 1) sharing only 70% homology with laboratory STB (type 2). Type 1 plasmids had a REP1 protein with 6-10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-microns variants that shared a similar STB consensus sequence exhibited a high degree of REP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution of trans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-microns variants into two main evolutionary lineages that differ at STB as well as REP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.

Unique composition of plastid chaperonin-60: alpha and beta polypeptide-encoding genes are highly divergent.

Molecular chaperones of the chaperonin family occur in prokaryotes and in plastids and mitochondria. Prokaryotic and mitochondrial chaperonin-60 oligomers (Cpn-60) are composed of a single subunit type (p60cpn-60). In contrast, preparations of purified plastid Cpn-60 contain approximately equal quantities of two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, with slightly different electrophoretic mobilities. We have isolated cDNA clones encoding plastid p60cpn-60 alpha and p60cpn-60 beta polypeptides from Brassica napus and Arabidopsis thaliana. The unexpected degree of sequence divergence observed between p60cpn-60 alpha and p60cpn-60 beta raises questions concerning the structure of the oligomer and the functions of these polypeptides. We have also found an amino acid sequence motif within all p60cpn-60 sequences which resembles the p10cpn-10 sequences.

Genetic variability in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase and its small subunit mRNA in pea.

The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was studied during greening in 12Pisum sativum L. genotypes. The proportion of mRNA coding for the small subunit of Rubisco (SSU mRNA) was also monitored by hybridization with a cDNA (complementary DNA) probe from one of the nuclear genes coding for SSU (rbcS). Both the SSU mRNA and Rubisco (in g·FW)(-1)) contents rapidly increased in all genotypes on illumination of dark-grown seedlings. Natural genetic variability was found in the amounts of SSU mRNA, Rubisco· (g FW)(-1), total RNA·(g FW)(-1) and mRNA·(µg total RNA)(-1). Differences among genotypes in SSU mRNA were apparently the result of differences in the rate of light-induced accumulation of therbcS gene transcripts. Genotype means for SSU mRNA and Rubisco· (g FW)(-1) amounts during greening were significantly correlated (r=0.788;P<0.01). This indicates a relationship between genetic differences in the rate of light-induced accumulation of therbcS gene transcripts and the Rubisco amount, and establishes a link between natural genetic variability at the molecular and the physiological levels. Genotypic variability in SSU mRNA during greening was also positively correlated to the Rubisco content per unit leaf area in fully greened leaves. Although Southern-blot analysis indicated that there was also natural genetic variability in the copy number of therbcS genes, this difference in copy number could not account for the differences in SSU mRNA production.

Site-specific cleavage of tobacco mosaic virus RNA: a study of factors influencing the cleavage.

DNA oligomer directed ribonuclease H (RNase H) methodology is applied to specifically cleave tobacco mosaic virus (TMV) RNA. Using a synthetic DNA oligomer P(dT8)dCdC, complementary to a region from nucleotide 5545 to nucleotide 5554 at the 3' end of TMV RNA, we have cleaved the RNA at the site of polynucleotides complementary to the DNA oligomer. Factors such as secondary structure of the RNA, concentrations of DNA oligomer, RNase H and magnesium ions in the reaction mixture, and time of incubation were optimized for the RNase H cleavage of TMV RNA-DNA oligomer complex. Denaturation of TMV RNA with 50% dimethyl sulphoxide at 50 degrees C is essential for the site-specific cleavage.

The effects of heterologous and homologous coat protein on alkaline disassembly of tobacco and tomato isolates of tobacco mosaic virus.

Tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) disassemble in dilute alkaline solutions (pH > 8.0). Tm-TMV is more sensitive to alkali than is Tb-TMV. Tb protein is, however, able to partially stabilize both Tm-TMV and Tb-TMV, thus retarding the rate of alkaline disassembly. Tm protein is unable to stabilize Tm-TMV and also increases the rate of alkaline disassembly of Tb-TMV.

A comparative study of the alkaline disassembly of two strains of tobacco mosaic virus.

Exposure of tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) to dilute alkaline solutions (pH > 8.0) at 0 degrees results in disassembly of the virus particles. Over the range of pH and NaCl concentration studied, Tm-TMV was more sensitive to alkaline conditions than was Tb-TMV. Kinetic analysis demonstrates that both isolates disassemble in a stepwise manner and that each produces six major intermediate-size particles.