DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: Analysis with mutated binding sites

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UAS_NTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5 or 3 sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

Pronase digestion of brush border membrane-bound Cry1Aa shows that almost the whole activated Cry1Aa molecule penetrates into the membrane

Bacillus thuringiensis insecticidal proteins, Cry toxins, following ingestion by insect larvae, induce insecticidal effect by penetrating the brush border membranes (BBM) of midgut epithelial cells. Purified, activated B. thuringiensis Cry1Aa bound to Bombyx mori BBMV or unbound Cry1Aa were vigorously digested with Pronase. Both digests were compared by Western blotting. Free Cry1Aa was digested to α-helix and/or to amino acids at 1 mg Pronase/mL within 2.4 h at 37 °C. Whereas, BBMV-bound Cry1Aa was very resistant to Pronase digestion and even at 2 mg for 24 h, 7.5 kDa and 30 kDa peptide were detected by α-2,3 antiserum, and α-4,5 and α-6,7 antisera, respectively. Another 30 kDa peptide was also detected by β-6-11 and domain III antisera. These fragments are believed either to be embedded in or to strongly interact with the BBMV. The 7.5 and former 30 kDa peptides are thought to be derived from α-2,3 helix and stretch of α-4 to α-7 helices. Furthermore the latter 30 kDa was thought to include the stretch of β-6 to domain III. Moreover, the embedded Cry1Aa molecule appears to be segregated in some areas of β-1-5 sheets, resulting in the above two 30 kDa peptides. From these digestion patterns, we proposed new membrane insertion model for single Cry1Aa molecule. On the other hand, in digestion of BBMV-bound Cry1Aa, 15 kDa peptide which was recognized only by α-4,5 antiserum was observed. This fragment must be dimeric α-4,5 helices and we discussed the origin of this peptide.     

The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination

In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cabI/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

DNA binding factor GT-2 from Arabidopsis

Complementary DNA clones encoding a DNA-binding factor have been obtained from Arabidopsis by DNA hybridization with a GT-2 factor cDNA clone from rice. The GT-2 gene appears to be present as a single copy in the Arabidopsis genome and is transcribed as a 2.1 kb mRNA which is not light-regulated. The longest open reading frame in the sequenced clones predicts a protein of 65 kDa, beginning with the first in-frame methionine. The protein contains basic, acidic, and proline/glutamine-rich motifs and has significant amino acid sequence homology to the rice GT-2 factor, including three regions of 50–75 amino acids each of greater than 60% identity. Two of these regions are predicted to form similar trihelix structures postulated to be involved in selective binding to specific variations of a GT-box motif DNA sequence found in the promoter regions of several plant genes. Except for weak similarity to a tobacco GT-box binding factor, GT-1a/B2F, Arabidopsis GT-2 has no similarity to other sequences in the databases. DNA-binding studies show that Arabidopsis GT-2 has binding characteristics similar to those of the rice GT-2 factor, but dissimilar to those of the tobacco GT-1a/B2F factor. The data indicate that a DNA-binding factor containing domains of similar structure and target-sequence specificity has been conserved between monocots and dicots.     

Light responsiveness of clock genes, Per1 and Per2, in the olfactory bulb of mice

The olfactory bulb (OB) of rodents has been suggested to possess a self-sustaining circadian oscillator which functions independent from the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. However, neither histology nor physiology of this extra-SCN clock is studied yet. In the present study, we examined circadian variation of major clock gene expressions in the OB and responsiveness to single photic stimuli. Here we show significant circadian variation in the expression of clock genes, Per1, Per2 and Bmal1 in the OB. Per1 and PER2 were mainly expressed in the mitral cell and granular cell layers of the OB. Light responsiveness of Per1 and Per2 expression was different in the OB from that in the parietal cortex. Both Per1 and Per2 are expressed in the OB only by l000 lux light pulse, whereas 100 lux light was enough to induce Per1 mRNA in the parietal cortex. Interestingly, even 1000 lux light failed to induce Per2 mRNA in the parietal cortex. These clock gene-specific and brain region-dependent responses to lights in the OB and parietal cortex suggest that single light stimulus induces various physiological functions in different brain areas via specific clock gene.     

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into and loci by 7 kb of noncoding sequence and are flanked by homologous genes -fg and -fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its locus, thus demonstrating that the compatible A mating interaction is between an HD1 and an HD2 protein. The A43 locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.     

Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution

The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event. Correspondence to: M. Pauly     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

RFLP patterns of gliadin alleles in Triticum aestivum L.: implications for analysis of the organization and evolution of complex loci

A correspondence between RFLP patterns and gliadin alleles at the Gli-1 and Gli-2 loci was established in a set of 70 common wheat (T.aestivum L.) cultivars using -gliadin (K32) and -gliadin (pTU1) specific probes. All Gli-B1 and Gli-D1 alleles which differed in encoded -gliadins showed definite RFLP patterns after hybridization with the K32 probe. Two groups of Gli-B1 alleles, Gli-B1b-like and Gli-B1e-like, were identified, and these could originate from distinct genotypes of the presumptive donor of the B-genome. Intralocus recombination and/or gene conversion as well as small deletions, gene silencing and gene amplification were assumed to be responsible for the origin of new gliadin alleles. Silent -gliadin sequences were shown to exist in all of the genotypes studied. K32 also differentiated Gli-A1a from all other Gli-A1 alleles as well as the Gli-B11 allele in cultivars carrying the 1B/1R (wheat/rye) translocation. PTU1 was shown to recognize several Gli-A2 alleles, but not the Gli-B2 or Gli-D2 alleles. Moreover, this probe hybridized to chromosome 1R sequences suggesting the existence of rye gene(s), probably silent, for -gliadin-like proteins on chromosome 1R.