Plant Molecular Genetics and Functional Genomics

ISII7, previously known as the 2.6 kb minicircle, is a transposable element found in Streptomyces coelicolor A3(2). It integrates predominantly into one preferred site when introduced into the closely related Streptomyces lividans 66, which lacks I S l l 7 . This preferred integration site was deleted from the S. lividans chromosome by replacement with an erythromycin resistance gene delivered by a q5C31 phage vector. When I S l l 7 was introduced into the resulting strain it integrated into many other sites, with some indication of site preference. By cloning a 200 bp fragment centred on the preferred integration site onto a low copy number, self-transmissible Streptomyces plasmid derived from SCP2* it was shown that this sequence is sufficient to define the preferred site: ISI17 integrates efficiently into this sequence from its preferred site in the host chromosome and at a lower frequency from the plasmid into the preferred site on the S. lividans chromosome.

Changes in telomere lengths and telomerase activity in tobacco cells were studied during dedierentiation and dierentiation; leaf tissues were used to initiate callus cultures, which were then induced to regenerate plants. While no signi®cant changes in the range of telomere lengths were observed in response to dedierentiation and dierentiation, there was a conspicuous increase in telomerase activity in calli compared to the source leaves, where the activity was hardly detectable. In leaves of regenerated plants, the telomerase activity fell to almost the same level as in the original plant, showing on the average 0.04% of the level in callus. The process was then repeated using the regenerants as the source material. In the second round of dedierentiation and dierentiation, telomerase activity showed a similar increase in calli derived from regenerated plants and a drop in plants regenerated from these calli. Telomere lengths remained unchanged both in calli and in leaves of regenerants. The conservation of telomere lengths over repeated rounds of dedierentiation and dierentiation, which are associated with dramatic changes in cell division rate and corresponding variation in telomerase activity may re¯ect the function of a regulatory mechanism in plant cells which controls telomerase action to compensate for replicative loss of telomeric DNA.

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 bp and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 bp and 160 bp KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 bp KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 bp KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 bp Kpnl families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 bp KpnI repeat probably evolved from the 160 bp Kpnl repeat since the missing 18 bp segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.

Climate change seriously impacts global agriculture, with rising temperatures directly affecting the yield. Vegetables are an essential part of daily human consumption and thus have importance among all agricultural crops. The human population is increasing daily, so there is a need for alternative ways which can be helpful in maximizing the harvestable yield of vegetables. The increase in temperature directly affects the plants' biochemical and molecular processes; having a significant impact on quality and yield. Breeding for climate-resilient crops with good yields takes a long time and lots of breeding efforts. However, with the advent of new omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, the efficiency and efficacy of unearthing information on pathways associated with high-temperature stress resilience has improved in many of the vegetable crops. Besides omics, the use of genomics-assisted breeding and new breeding approaches such as gene editing and speed breeding allow creation of modern vegetable cultivars that are more resilient to high temperatures. Collectively, these approaches will shorten the time to create and release novel vegetable varieties to meet growing demands for productivity and quality. This review discusses the effects of heat stress on vegetables and highlights recent research with a focus on how omics and genome editing can produce temperature-resilient vegetables more efficiently and faster.

or F3′H promoter sequences resulted in the regeneration of 6 and 4 transgenic lines, respectively. A complete red coloration of all plant organs was found in four out of six transgenic lines harboring the 35S-MdMYB10-GFP43 construct. Less red coloration of plant organs was found for lines transformed with the F3′H-MdMYB10-GFP43 construct. The MdMYB10 gene shows only limited suitability as a reporter gene for promoter studies in strawberries because weak promoter activity is difficult to distinguish, particularly in tissues showing a strongly colored background such as green leaves. GFP specific fluorescence signals were detectable neither in tissue strongly expressing MdMYB10 nor in green tissue of any transgenic line. The reason for this remained unclear but it can be excluded that it was due to incorrect splicing. • Reporter gene • CaMV 35S promoter • Green fluorescent protein (GFP) Abbreviations CDS Coding sequence CH3H Chalcone 3-hydroxylase CaMV Cauliflower mosaic virus Cy equ Cyanidin equivalent F3′H Flavonoid 3′-hydroxylase GFP Green fluorescent protein Md Malus domestica Communicated by Wenwu Guo.

Target of rapamycin (TOR) is a conserved central growth regulator in eukaryotes that has a key role in maintaining cellular nutrient and energy status. Arbuscular mycorrhizal (AM) fungi are mutualistic symbionts that assist the plant in increasing nutrient absorption from the rhizosphere. However, the role of legume TOR in AM fungal symbiosis development has not been investigated. In this study, we examined the function of legume TOR in the development and formation of AM fungal symbiosis. RNA-interference-mediated knockdown of TOR transcripts in common bean (Phaseolus vulgaris) hairy roots notably suppressed AM fungus-induced lateral root formation by altering the expression of root meristem regulatory genes, i.e., UPB1, RGFs, and sulfur assimilation and S-phase genes. Mycorrhized PvTOR-knockdown roots had significantly more extraradical hyphae and hyphopodia than the control (empty vector) roots. Strong promoter activity of PvTOR was observed at the site of hyphal penetration and ...

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualiWes to be the cis-acting NRE for all the responsive genes. We searched for the speciWc occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-speciWc bias. This is the Wrst comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any speciWcity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-speciWc and shoot-speciWc response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants.

The inheritance of flowering time (FT) in a cross between early-flowering (P1) and late-flowering (P2) Matthiola incana genotypes, was investigated . The distribution of FT in F1, F2 and BC1 generations indicated an additive genetic control with partial dominance of earliness alleles, particularly with regard to double-flowered plants . Single-flowered plants flowered earlier than double-flowered plants, averaged within both parental lines ; however, the mean difference between singles and doubles was 16 days for P1 as compared with 6 days for P2 . This flowerdoubleness related delay in FT was found to be heritable, by analysis of F3 family means . The implications of these results on the breeding of stock cultivars is discussed .

DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5' terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3' terminus. Increasing annealing temperatures from 15 ° to 70 ° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3'terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The DSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up-or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from DSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500fold down-regulated in the DSmta-1 mutant while in all other sterile mutants this gene is up-regulated.

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage.

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage.

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targ...

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in revl and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

We recently described the ABC transporter Ngc (encoded by the ncgEFG operon) from Streptomyces olivaceoviridis, the first of its kind to be shown to transport N-acetylglucosamine and N,N'-diacetylchitobiose (chitobiose). A chromosomal mutant carrying a disruption of the ngcE gene, which encodes the sugar binding protein, was still able to transport N-acetylglucosamine. This phenotype can now be attributed to a functional phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two adjacent homologous genes, ptsC1 and ptsC2, were identified, and deduced to encode proteins which are 56% identical and can be predicted to contain eight transmembrane regions. PtsC1 (432 amino acids) and PtsC2 (403 residues) each correspond to a single EIIC domain; such domains are otherwise known only in several bacterial multidomain permeases for glucose/mannose or N-acetylglucosamine. The C-terminal sequences of PtsC1 and PtsC2 correspond to the motifs LKTPGREP and LPTR-GRES, respectively. The ptsB gene located upstream of ptsC1 is predicted to encode a homologue of the EIIB domains usually found in bacterial multidomain permeases. Physiological and biochemical analyses of ngcE mutants carrying disruptive insertions in ptsC1 or ptsC2 or both revealed that, when grown on N-acetylglucosamine, the membrane component PtsC2, unlike PtsC1, mediates PEP-dependent, specific (K m =5 lM) transport of N-acetylglucosamine, but not of other hexoses. Crosscomplementation of membrane and cytoplasmic fractions from the various mutants led to the conclusion that S. olivaceoviridis also expresses the functional soluble components HPr, EI and EIIA of the PTS system. During growth on xylose, uptake of this pentose occurred if ptsC1 or ptsC2 was intact, but not in a mutant containing disrupted forms of both genes.

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.

To understand the mechanisms of aluminum (Al) tolerance in wheat (Triticum aestivum L.), suppression subtractive hybridization (SSH) libraries were constructed from Al-stressed roots of two nearisogenic lines (NILs). A total of 1,065 putative genes from the SSH libraries was printed in a cDNA array. Relative expression levels of those genes were compared between two NILs at seven time points of Al stress from 15 min to 7 days. Fifty-seven genes were diVerentially expressed for at least one time point of Al treatment. Among them, 28 genes including genes for aluminum-activated malate transporter-1, ent-kaurenoic acid oxidase-1, -glucosidase, lectin, histidine kinase, and phospoenolpyruvate carboxylase showed more abundant transcripts in Chisholm-T and therefore may facilitate Al tolerance. In addition, a set of genes related to senescence and starvation of nitrogen, iron, and sulfur, such as copper chaperone homolog, nitrogen regulatory gene-2, yellow stripe-1, and methylthioribose kinase, was highly expressed in Chisholm-S under Al stress. The results suggest that Al tolerance may be co-regulated by multiple genes with diverse functions, and those genes abundantly expressed in Chisholm-T may play important roles in enhancing Al tolerance. The down-regulated genes in Chisholm-S may repress root growth and restrict uptake of essential nutrient elements, and lead to root senescence.

Most commercially produced cotton cultivars have two types of fibers on the seed coat, short fuzz and long lint. Lint fiber is used in the textile industry, while fuzz is considered an undesirable trait. Both types of fibers are believed to be controlled by the same regulators; however, their mechanisms of actions are still obscure. Cotton fiber mutants provide an excellent system to study the genes that regulate fiber development. Here we described four uncharacterized and three previously reported cotton mutants with fuzzless seed phenotypes. To evaluate whether or not the genes previously associated with fuzzless seed phenotypes have mutations we sequenced whole genomic DNA of seven mutants and wild type varieties. We identified multiple polymorphic changes among the tested genes. Non-synonymous SNPs in the coding region of the MML3-A gene was common in the six mutant lines tested in this study, showing both dominant and recessive fuzzless phenotypes. We have mapped the locus of the causative mutation for one of the uncharacterized fuzzless lines using an F 2 population that originated from a cross between the dominant fuzzless mutant and a wild type. Further, we have clarified the current knowledge about the causative n 2 mutations by analyzing the sequence data and previously reported mapping data. The key genes and possible mechanisms of fiber differentiation are discussed in this study. Gossypium hirsutum • Cotton fiber • Fuzzless • Fiberless • Single nucleotide polymorphism * Marina Naoumkina

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, "Gene Regulatory Networks and Network Models in Development and Evolution," held April 12-14, 2016, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at www.nasonline.org/ Gene_Regulatory_Networks.

Three genes for the subunits of the NADH dehydrogenase (nadS, had4, and nad2) are tandemly clustered on the liverwort mitochondrial genome. Their gene products showed high levels of amino acid sequence identity with the corresponding subunits from higher plant mitochondria (82.8-84.4%), and significant levels of identity with those from liverwort chloroplast (32.0-33.5%), Podospora anserina mitochondria (21.4-45.9 %), and human mitochondria (18.4-27.9 %). In addition, these three subunits from liverwort mitochondria have conserved amino acid residues in their central regions. The gene nad5 is interrupted by a 672. bp group I intron, while genes nad4 and nad2 are interrupted by group II introns of 899 bp and 1418 bp, respectively. Northern blot analysis using exon-intron specific probes indicated that these three genes are transcribed as a single precursor mRNA of 9.6 kb in length and are processed into mature mRNA molecules in liverwort mitochondria. Several regions of this nad gene cluster are repeated in the liverwort mitochondrial genome.

The Class V zygote-specific gene from Chlamydomonas reinhardtii has been cloned and sequenced. This gene encodes a polypeptide of 86 amino acids, which contains a signal peptide and 6 cysteine residues arranged in an inverted symmetrical repeat. The Class V gene product is postulated to be a component of the zygote cell wall. Southern analysis revealed two tandemly oriented and closely linked copies of the Class V gene, designated A and B. The A gene appears to be a pseudogene, based on analysis of Class V cDNAs, primer extension with gene-specific primers, and Northern analysis which failed to detect an A gene transcript. Genetic analysis using a related Chlamydomonas species that lacks the A gene, but which produces normal zygotic progeny, further indicates that the A gene is not required for zygote development.

Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.

In about 30% of infantile, juvenile, or adolescent patients with steroid-resistant nephrotic syndrome (SRNS), a monogenic cause can be identified. The histological finding in SRNS is often focal segmental glomerulosclerosis (FSGS). Genetic data on adult patients are scarce with low diagnostic yields. Exome sequencing (ES) was performed in patients with adult disease onset and a high likelihood for hereditary FSGS. A high likelihood was defined if at least one of the following criteria was present: absence of a secondary cause, ≤25 years of age at initial manifestation, kidney biopsy with suspicion of a hereditary cause, extrarenal manifestations, and/or positive familial history/reported consanguinity. Patients were excluded if age at disease onset was <18 years. In 7/24 index patients with adult disease onset, a disease-causing variant could be identified by ES leading to a diagnostic yield of 29%. Eight different variants were identified in six known genes associated with monog...

is study aimed at developing a suitable and reproducible protocol for in vitro micropropagation of Aloe adigratana Reynolds using explants from offshoots with the help of the most commonly used plant growth regulators (PGRs). Explants were initiated in full-strength Murashige and Skoog (MS) media enriched with 0.2 mg/L benzylaminopurine (BAP) + 0.2 mg/L naphthaleneacetic acid (NAA). Shooting experiment was conducted in full-strength MS media enriched with 0/0, 0.5/0.5, 1.0/0.5, 1.5/0.5, and 2.0/ 0.5 mg/L BAP/NAA. Likewise, rooting experiment was carried out in half-strength MS media enriched with NAA at 0.5, 1.0, and 1.5 mg/L and indol-3-butyric acid (IBA) at 0.5, 1.0, and 1.5 mg/L. Finally, acclimatization study was conducted in greenhouse, nursery, and open field. e shooting response of the plant was best in MS media enriched with 1.0 mg/L BAP + 0.5 mg/L NAA. is media formulation resulted in the shortest mean number of days to shooting (14.00 ± 2.30 days), the highest mean shoot number (4.00 ± 3.40), and the second highest mean shoot length (8.60 ± 2.10 cm). IBA enhanced rooting at higher concentrations (1.0 and 1.5 mg/L), but NAA did the same at lower concentrations (0.5 and 1.0 mg/L). All plantlets (n � 62) survived primary acclimatization. Secondary acclimatization in composted and matured soil media under nursery and open field (sun light) condition produced 88 to 93% survival rate. e death of plantlets in the secondary acclimatization is accounted to mechanical damage. is study demonstrated that the tested PGRs were useful in enhancing the in vitro micropropagation of the plant. Future studies need to focus on optimizing the protocol for large-scale, commercial micropropagation.

A method for selection of constitutive cysB mutation is described which takes advantage of the resistance of cysteine constitutive mutants to 1,2,4-triazole. Since cysM cysK double mutants are cysteine auxotrophs, by selecting for triazole resistance in cysM strains, mutants arising under this condition also should be constitutive for cysteine biosynthesis. Genetic analysis of mutants isolated by this technique showed that their mutational sites are located in the cysB region. Biochemical assays of cysteine enzymes, sulphite reductase and O-acetylserine sulfhydrylase of the mutants showed the derepressed level of these enzymes and the lack or slight repression by 1-cysteine.

We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MEG) or an Or-n-butyl-guanine (BUG) paired with C at the first position of the third codon in gene G of bacteriophage ~X174 form I' DNA . We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C :MeG or C :BUG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T: BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia eoli ABlI57) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contrast to this, the transition frequency from T:MeG is high (5_+2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process. Using the BuG data reported here and

The genes janus (jan) A and B, and serendipity (sty) fl and 6 are two pairs of duplicated genes that are adjacent to each other on the third chromosome of Drosophila melanogaster. The jan A and sry fl genes are expressed throughout development in both males and females. They are transcribed in opposite orientations from start sites separated by only 173 bp of DNA. We report here the complete sequence of the jan A and B genes in Drosophila pseudoobscura, a species distantly related to D. melanogaster in which the overall organization of the sry ~, jan A and jan B genes is identical to that in D. melanogaster. Sequence comparison of the jan A-sry ~ intergenic region and 5'-transcribed domain of each gene between D. melanogaster and D. pseudoobscura reveals short stretches of conserved sequences that may correspond to cis-acting regulator elements. In order to test the possibility that some cis-acting regulatory sequences are shared by the two genes, we carried out a deletion analysis of the jan A/sry ~ intergenic region in D. melanogaster using transgenic lacZ fusion genes. Our results show that sry fl cis-acting sequences are located in the (-117; + 137) 5'-region of the gene and that jan A cis-regulatory sequences are included in the (-56; +151) 5'-domain of this gene. Together these data indicate that in spite of the physical proximity of the jan A and sry fl genes, their transcription is regulated by separate cis-acting sequences.

This study aimed to quantify the influence of the astrocyte proximity on myelination genomic fabric (MYE) of oligodendrocytes, defined as the most interconnected and stably expressed gene web responsible for myelination. Such quantitation is important to evaluate whether astrocyte signaling may contribute to demyelination when impaired and remyelination when properly restored. For this, we compared changes in the gene expression profiles of immortalized precursor oligodendrocytes (Oli-neu), stimulated to differentiate by the proximity of nontouching astrocytes or treatment with db-cAMP. In a previous paper, we reported that the astrocyte proximity upregulated or turned-on a large number of myelination genes and substantially enriched the Ca 2? -signaling and cytokine receptor regulatory networks of MYE in Oli-neu cells. Here, we introduce the ''transcriptomic distance'' to evaluate fabric remodeling and ''pair-wise relevance'' to identify the most influential gene pairs. Together with the prominence gene analysis used to select and rank the fabric genes, these novel analytical tools provide a comprehensively quantitative view of the physio/pathological transformations of the transcriptomic programs of myelinating cells. Applied to our data, the analyses revealed not only that the astrocyte neighborhood is a substantially more powerful regulator of myelination than the differentiating treatment but also the molecular mechanisms of the two differentiating paradigms are different. By inducing a profound remodeling of MYE and regulatory transcriptomic networks, the astrocyte-oligodendrocyte intercommunication may be considered as a major player in both pathophysiology and therapy of neurodegenerative diseases related to myelination.

We present here the de novo genome assembly CerEla1.0 for the red deer, Cervus elaphus, an emblematic member of the natural megafauna of the Northern Hemisphere. Humans spread the species in the South. Today, the red deer is also a farm-bred animal and is becoming a model animal in biomedical and population studies. Stag DNA was sequenced at 74× coverage by Illumina technology. The ALLPATHS-LG assembly of the reads resulted in 34.7 × 10scaffolds, 26.1 × 10of which were utilized in Cer.Ela1.0. The assembly spans 3.4 Gbp. For building the red deer pseudochromosomes, a pre-established genetic map was used for main anchor points. A nearly complete co-linearity was found between the mapmarker sequences of the deer genetic map and the order and orientation of the orthologous sequences in the syntenic bovine regions. Syntenies were also conserved at the in-scaffold level. The cM distances corresponded to 1.34 Mbp uniformly along the deer genome. Chromosomal rearrangements between deer and ...

In the era of the diseasomes and interactome networks, linking genetics with phenotypic traits in Turner syndrome should be studied thoroughly. As a part of this stratagem, mosaicism of both X and Y chromosome which is a common finding in TS and an evaluation of congenital heart diseases in the different situations of mosaic TS types, can be helpful in the identification of disturbed sex chromosomes, genes and signaling pathway actors. Here we report the case of a mosaic TS associated to four left-sided CHD, including BAV, COA, aortic aneurysms and dissections at an early age. The mosaicism included two cell lines, well-defined at the cytogenetic and molecular levels: a cell line which is monosomic for Xp and Xq genes (45,X) and another which is trisomic for pseudoautosomal genes that are present on the X and Y chromosomes and escape X inactivation: 45,X[8]/46,X,idic(Y)(pter→q11.2::q11.2→pter) [42]. This case generates two hypotheses about the contribution of genes linked to the sex chromosomes and the signaling pathways involving these genes, in left-sided heart diseases. The first hypothesis suggests the interaction between X chromosome and autosomal genes or loci of aortic development, possibly dose-dependent, and which could be in the framework of TGF-β-SMAD signaling pathways. The second implies that left-sided congenital heart lesions involve sex chromosomes loci. The reduced dosage of X chromosome gene(s), escaping X inactivation during development, contributes to this type of CHD. Regarding our case, these X chromosome genes may have homologues at the Y chromosome, but the process of inactivation of the centromeres of the isodicentric Y spreads to the concerned Y chromosome genes. Therefore, this case emerges as an invitation to consider the mosaics of Turner syndrome and to study their phenotypes in correlation with their genotypes to discover the underlying developmental and genetic mechanisms, especially the ones related to sex chromosomes. Aortic coarctation • Acute type A aortic dissection • Bicuspid aortic valve • Congenital heart diseases • Isodicentric (Yp) • Turner syndrome Abbreviations AAAD Acute type A aortic dissection ACTA2 Actin, alpha-2, smooth muscle, aorta [MIM:102620] cytogenetic location:10q23.31 AGTR1 Angiotensin II receptor type 1 [MIM:106165] cytogenetic location:3q24 AGTR2 Angiotensin II receptor type 2 [MIM:300034] cytogenetic location:Xq23 AngII Angiotensin II AS Aortic stenosis AZF Azoospermia factor BAV Bicuspid aortic valve bp Base pair Communicated by S. Hohmann.

The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22)is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hyIA from the 5' end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.

The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable ill E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca 2 + for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca 2 + concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca 2 + concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca 2+. These data indicate that the repeat domain of haemolysin is responsible for Ca 2 +-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca 2 + in haemolysis is presented.

The genesis of small repeats involved in infrequent recombinations in plant mitochondrial genomes remains unclear. We propose that at least some of the small repeats are generated in a similar way to the large, highly recombinogenic, plant mitochondrial repeats. A 314-bp sequence was detected as a small, rarely recombining mitochondrial repeat in the genus Phaseolus. Two of the recombinational forms were predominant, while two others were found in substoichiometric amounts in the species P. vulgaris, P. polyanthus and P. coccineus. However, the pairs of predominant and substoichiometric forms were distinct in each genome, indicating that a mechanism other than recombination is responsible for their maintenance in high or low copy number. In P. lunatus, which is phylogenetically quite remote from the other species examined, only one form of the 314-bp repeat was predominant, while the other forms were present in substoichiometric amounts. In this genome, we also identi®ed sequences containing the terminal 11 or 7 bp of the 314-bp repeat. These con®gurations could serve as intermediates during generation of the repeat. We presume that two site-speci®c recombinations between the intermediates and the predominant form found in P. lunatus resulted in creation of the two new forms of the repeat. The fourth form of the repeat appeared after a further recombination that occurred at the substoichiometric level. The nature of this recombination, whether site-speci®c or homologous, is discussed. Beyond the evident similarities between the model presented in this work and the three-recombination models previously proposed to explain the formation of large, frequently recombining repeats, we did not detect any speci®c deletion associated with generation of the repeat.

We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25 S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.

The constitutive expression of an antisense chalcone synthase (CHS) gene in transgenic petunia plants results with high frequency in a reduced flower pigmentation due to a reduction in the CHS mRNA steady-state level in floral tissue. Here we show that this reduction is specific for CHS mRNA; chalcone flavanone isomerase (CHI) and dihydroflavonol reductase (DFR) mRNA steady-state levels are unaffected. However, in white floral tissue a severe reduction in CHI specific activity is found, accompanied by an altered signal for CHI protein on western blots. We find no correlation between the phenotypic effect of the antisense CHS gene and its chromosomal position. For some of the antisense CHS transformants the flower phenotype is highly variable. We demonstrate that pigmentation in these plants can be influenced by gibberellic acid and light, suggesting that the variable flower phenotype is caused by changes in physiological conditions during flower development. The results not only indicate that flower pigmentation in these plants reveals the variable expression of the antisense transgene, but also show that genomic sequences flanking the transgene may render its expression extremely susceptible to physiological conditions.

Food, horticulture, agriculture, and food science are among the industries that are using nanotechnology more and more to preserve fruits and vegetables. Cost and inadequate shelf-life improvement are two disadvantages of conventional approaches. By extending these products' shelf lives, nanotechnology can reduce manufacturing and postharvest losses. Because of their special properties, nanoparticles are important in food packaging and storage as they improve food quality and lower the rates at which oxygen, water vapour, and relative humidity are transmitted.

Background: Wound healing is a representative phenomenon of potato tubers subjected to mechanical injuries. Our previous results found that benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) promoted the wound healing of potato tubers. However, the molecular mechanism related to inducible wound healing remains unknown. Results: Transcriptomic evaluation of healing tissues from potato tubers at three stages, namely, 0 d (nonhealing), 5 d (wounded tubers healed for 5 d) and 5 d (BTH-treated tubers healed for 5 d) using RNA-Seq and differentially expressed genes (DEGs) analysis showed that more than 515 million high-quality reads were generated and a total of 7665 DEGs were enriched, and 16 of these DEGs were selected by qRT-PCR analysis to further confirm the RNA sequencing data. Gene ontology (GO) enrichment analysis indicated that the most highly DEGs were involved in metabolic and cellular processes, and KEGG enrichment analysis indicated that a large number of DEGs were associated with plant hormones, starch and sugar metabolism, fatty acid metabolism, phenylpropanoid biosynthesis and terpenoid skeleton biosynthesis. Furthermore, a few candidate transcription factors, including MYB, NAC and WRKY, and genes related to Ca 2+ -mediated signal transduction were also found to be differentially expressed during wound healing. Most of these enriched DEGs were upregulated after BTH treatment. This comparative expression profile provided useful resources for studies of the molecular mechanism via these promising candidates involved in natural or elicitor-induced wound healing in potato tubers.

The results of short interrupted matings between an Hfr donor and a recipient strain carrying a temperature-sensitive replication mutant (frp) of Flac demonstrate that the Hfr strain transfers this frp gene off early in conjugation. This frp gene was also shown to function in the maintenance of mutant F' plasmids which appear to be generated from the DNA transferred early in conjugation by Hfr donors. In the course of these experiments, it was further demonstrated that certain Hfr strains which had been described as transferring the tra genes early in fact transfer that region of F late in conjugation. ,Z3

A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-IO1. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEpl3 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-IO1 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5' end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.

RNA polymerase II (Pol II) is composed of a ten subunit core and a two subunit dissociable subcomplex comprising the fourth and seventh largest subunits, RPB4 and RPB7. The evolutionary highly conserved RPB4/7 heterodimer is positioned in the Pol II such that it can make contact with various factors involved in RNA biogenesis and is believed to play roles both during the process of transcription and post-transcription. A detailed analysis of RPB4/ 7 function in a multicellular eukaryote, however, is lacking partly because of the lack of a suitable genetic system. Here, we describe generation and initial analysis of Drosophila Rpb4 mutants. In the fly, RPB4 is a product of a bicistronic gene together with the ATAC histone acetyltransferase complex constituent ADA2a. DmAda2a and DmRpb4 are expressed during fly development at different levels. The structure of mature mRNA forms suggests that the production of DmADA2a and DmRPB4-specific mRNAs is ensured by alternative splicing. Genetic analysis indicates that both DmRPB4 and DmADA2a play essential roles, because their absence results in lethality in early and late larval stages, respectively. Upon stress of high temperature or nutritional starvation, the levels of RPB4 and ADA2a messages change differently. RPB4 colocalizes with Pol II to several sites on polytene chromosomes, however, at selected locus, the abundances of Pol II and RPB4 vary greatly. Our data suggest no tight functional link between DmADA2a and DmRPB4, and reveal differences in the abundances of Pol II core subunits and RPB4 localized at specific regions on polytene chromosomes, supporting the suggested role of RPB4 outside of transcription-engaged Pol II complexes.

The function and the molecular properties of the Rpt1/p48B ATPase subunit of the regulatory particle of the Drosophila melanogaster 26S proteasome have been studied by analyzing three mutant Drosophila stocks in which P-element insertions occurred in the 5Ј-non-translated region of the Rpt1/p48B gene. These P-element insertions resulted in larval lethality during the second instar larval phase. Since the Rpt1/p48B gene resides within a long intron of an annotated, but uncharacterized Drosophila gene (CG17985), the second instar larval lethality may be a consequence of a combined damage to two independent genes. To analyze the phenotypic eVect of the mutations aVecting the Rpt1/p48B gene alone, imprecise P-element excision mutants were selected. One of them, the pupal lethal P1 mutation, is a hypomorphic allele of the Rpt1/ p48B gene, in which the displacement of two essential regulatory sequences of the gene occurred due to the insertion of a 32 bp residual P-element sequence. This mutation caused a 30-fold drop in the cellular concentration of the Rpt1/p48B mRNA. The decline in the cellular Rpt1/p48B protein concentration induced serious damage in the assembly of the 26S proteasomes, the accumulation of multiubiquitinated proteins, a change in the phosphorylation pattern of the subunit and depletion of this ATPase protein from the chromatin.

The excessive amounts of nitrogen applied in current farming systems can cause environmental problems. There is therefore a need to improve the ability of crop plants to utilize nitrogenous fertilizers. We screened for nitrogen deficiency-tolerant lines among transgenic rice plants that overexpressed full-length cDNAs (FL-cDNAs) corresponding to low-nitrogen response genes, genes related to nitrogen metabolism, and genes related to carbon metabolism. We found that overexpression of OsCPK12 FL-cDNA, encoding a calcium-dependent protein kinase (CDPK), conferred tolerance to lownitrogen stress in rice. After two weeks of low-nitrogen treatment, dry weights of shoots from OsCPK12-overexpressing plants were greater than those from control plants. Furthermore, total nitrogen contents of OsCPK12-overexpressing plants were higher than those of the control plants. Our findings suggest that OsCPK12 is involved in the signal transduction pathway(s) in the low-nitrogen stress response and may be useful in engineering crop plants with improved tolerance to low nitrogen levels.

Fusarium head blight (FHB), caused by the fungus Fusarium graminearum, represents one of the major wheat diseases worldwide, determining severe yield losses and reduction of grain quality due to the accumulation of mycotoxins. The molecular response associated with the wheat 2DL FHB resistance QTL was mined through a comprehensive transcriptomic analysis of the early response to F. graminearum infection, at 3 days post-inoculation, in spikelets and rachis. The analyses were conducted on two near isogenic lines (NILs) differing for the presence of the 2DL QTL (2-2618, resistant 2DL+ and 2-2890, susceptible null). The general response to fungal infection in terms of mRNAs accumulation trend was similar in both NILs, even though involving an higher number of DEGs in the susceptible NIL, and included down-regulation of the primary and energy metabolism, up-regulation of enzymes implicated in lignin and phenylpropanoid biosynthesis, activation of hormons biosynthesis and signal transduction pathways and genes involved in redox homeostasis and transcriptional regulation. The search for candidate genes with expression profiles associated with the 2DL QTL for FHB resistance led to the discovery of processes differentially modulated in the R and S NILs related to cell wall metabolism, sugar and JA signaling, signal reception and transduction, regulation of the redox status and transcription factors. Wheat FHB response-related miRNAs differentially regulated were also identified as putatively implicated in the superoxide dismutase activities and affecting genes regulating responses to biotic/abiotic stresses and auxin signaling. Altered gene expression was also observed for fungal non-codingRNAs. The putative targets of two of these were represented by the wheat gene WIR1A, involved in resistance response, and a gene encoding a jacalin-related lectin protein, which participate in biotic and abiotic stress response, supporting the presence of a cross-talk between the plant and the fungus.

The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes. The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome. (mtDNA) of the somatic hybrids described by showed an extensive rearrangement of somatic hybrid mtDNAs relative to mtDNAs of parental lines 3704 and 3688 . By examining the relationship between restriction banding patterns and CMS expression, identified a CMS-specific arrangement of mtDNA present only in sterile somatic hybrids and the sterile parent. An analysis of the relationship between CMS and cpDNA composition was not performed on these somatic hybrids, since no differences could be detected in the cpDNA restriction patterns of the parental lines. Further examination of cpDNA restriction patterns of the two parental lines used in these studies, 3704 and 3688, showed a difference in the mobility of a single EcoRl fragment. Presented here are the results of an analysis of the chloroplast DNA composition of six plants from the original set of Petunia somatic hybrids as well as the results from two additional sets of somatic hybrids. One of these sets was derived from the fusion of fertile P. parodii line 3699 and CMS P. hybrida line 2423; the other was derived from fusion of fertile P. hybrida line 3677 and CMS P. parodii line 3688.

The AciDs transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S: :TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S: :TPase produced many streptomycin-resistant (strep r) progeny, but in many of these families a small proportion of strep r seedlings were also resistant to hygromycin (hygr). Nevertheless, 70% of families tested did give rise to at least one strep r, hyg r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep~hyg ~ progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep r seedlings were also hyg ~ than after activation by 35S: :TPase. We also examined the genotype of strep ~, hyg ~ seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S: :TPase usually occur prior to floral development, and that although a low proportion of strep r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.

Glucose represses mitochondrial biogenesis and the fermentation of maltose, galactose and sucrose in yeast. We have analyzed the effect of D-glucosamine on these functions in order to determine if it can produce a similar repression. It was found that glucosamine represses the respiration rate (QO2) but more rapidly than glucose and to a final level slightly higher than in glucose-treated cells. Derepression of the respiration rate following either glucose or glucosamine repression was similar. A two hour lag was followed by a linear increase in QO 2 to the derepressed level. Both glucose and glucosamine repressed the level of cytochrome oxidase to the same level. Glucosamine was also found to repress maltose and galactose fermentation but not sucrose fermentation. The derepression of maltase synthesis was inhibited by glucosamine. The constitutive synthesis of maltase was repressed by the addition of glucosamine. Glucosamine was judged to produce a repressed state similar to glucose repression in many respects.

characterized two complementing functions required for maltose fermentation: MALp and MALg. The presence of either function alone is not sufficient to allow for the fermentation of maltose but diploids heterozygous for these loci were able to ferment. Using MALp, MALg and real ° tester strains obtained from Naumov (a real ° strain is a non-fermenting strain which does not complement either MALp or MALg strains), we have performed a genetic: analysis on the maltose fermenting strains 4059 (MALl) and 1412-4D (MAL3). These strains were shown to contain both the dominant MALl or MAL3 locus and an additional cryptic MALg. The presence of the dominant MALl or MAL3 locus alone is sufficient to enable the strain to ferment maltose and thus must contain both the MALp and MALg functions. The expression of the cryptic MALg in each strain must therefore be dependent upon the MALp function linked to the dominant locus. In 4059, the cryptic MALg locus is linked to the MAL3 locus and is thus denoted MAL3g. In 1412-4D, the cryptic MALg locus is linked to the MALl locus and is thus denoted MALlg. Gel transfer analysis of these strains was able to detect genomic HindIII fragment linked to both the dominant MAL loci and the MALg loci using a probe derived from the MAL6 locus of the Saceharomyces strain CBll. The significance of this result with regard to the genome organization of the MAL loci is discussed.

The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, ga13(2) strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive. The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlDpgl deletions were isolated from gaF331 (a revertant of gal3 which retains IS2) and transferred to 2gaI genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gaF200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ~ mutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage 2 contributes to the specificity and, in some instances, the frequency of IS2specific deletions. A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the For offprints contact." Asad Ahmed insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.