Isolation and chromosomal localization of ecdysterone-responsive genes in a Drosophila cell line

Cultured Kc 0% cells of Drosophila melanogaster are responsive to ecdysterone treatment. A library of lambda phages carrying segments of Drosophila genomic DNA was differential screened using poly(A)⁺RNAs from control and ecdysterone-treated cells. Nine independent recombinant phages that hybridized more intensely with poly(A)⁺ RNA from treated cells and six that hybridized most strongly with poly(A)⁺RNA from untreated cells were selected. Genomic localization of these inducible and repressible sequences was determined by hybridization in situ. These results suggest that expression of several unique genes is increased by the hormone. The six repressible sequences each contained DNA that hybridized to multiple chromosomal sites and appeared to be mobile elements, suggesting that the steroid hormone might be acting on the transposable elements. These probes will be useful for the study of positive and negative steroid regulation within the same cell.     

The zygomycetous fungus,Benjaminiella poitrasii contains a large family of differentially regulated chitin synthase genes

Benjaminiella poitrasii is a zygomycetous, non-pathogenic dimorphic fungus. Chitin synthases are the membrane bound enzymes involved in the synthesis of chitin and are key enzymes in the cell wall metabolism. Multiplicity of these enzymes is a common occurrence. Here, we identify eight distinct CHS genes in B. poitrasii as confirmed through DNA sequence and Southern analysis. These genes are related to other fungal CHS genes. BpCHS1-4 are class I-III chitin synthases while BpCHS5-8 are class IV-V chitin synthases. These eight genes are differentially expressed during morphogenesis and under different growth conditions. Two of these genes viz. BpCHS2 and BpCHS3 appear to be specific to the mycelial growth form. These are the first B. poitrasii sequences to be reported. Based on CHS gene sequences, B. poitrasii chitin synthase genes place it with other zygomycetes on a fungal phylogenetic tree.     

The active site of yeast endo-polygalacturonase contains seven subsites

The rate of hydrolysis of oligomers by the endopolygalacturonase of yeast is in the order: heptamer > hexamer > pentamer > tetramer. This suggests that the active site accommodates at least 7 units. Since the heptamer disappears concurrently with the bulk of larger oligomers, the maximum number of units appears to be 7. The release of labelled (unsaturated, or ³H labelled and reduced) end units from larger substrate is interpreted to indicate that the enzyme interacts with 3 saccharide units toward the reducing end from the bond to be broken, and with 4 units toward the non-reducing end. The relative affinities for the enzyme of saccharide units in various positions are unequal, as indicated by the very low relative rate of monomer production from the hydrolysis of hexamer and pentamer, and the apparently unequal probability of two other modes of hexamer hydrolysis [(tetramer + dimer) = 2.5 (trimer + trimer)].     

Nucleotide variation of seven genes in Drosophila kikkawai

We examined levels and patterns of nucleotide variation in 21 strains of Drosophila kikkawai from Miyako island, Japan for the partial regions of the following seven nuclear genes: Adh, Ddc, esc, ksr, Pgi, su(f), and Tpi. The nucleotide variation at total sites (pi(t)) ranged from 0.0013 in the ksr, to 0.0173 in the Adh. The nucleotide divergence at total sites (K(t)) between D. kikkawai and D. lini ranged from 0.0286 in the Tpi to 0.0687 in the su(f). The levels of nucleotide polymorphism and divergence were heterogeneous among the investigated gene regions. The HKA test, which tests imbalance between the intra and interspecific nucleotide variation, showed that the intraspecific nucleotide variation in the Pgi region was much lower than the interspecific variation, while intraspecific variation in the Tpi region was only slightly lower than interspecific variation. The MK test showed an excess of low frequency replacement polymorphic changes in the Adh region, suggesting that most replacement mutations are deleterious. Fay and Wu's test detected an excess of newly arisen variants in the Ddc region. In total, four of the seven gene regions showed significant deviation from the neutrality.     

The active site of Serratia endonuclease contains a conserved magnesium-water cluster.

Serratia endonuclease is an important member of a class of magnesium dependent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site of this enzyme. The sole protein ligand of the magnesium atom is Asn119; this metal ion is also associated with five water molecules to complete an octahedral coordination complex. These water molecules are very well ordered and there is no evidence of rotational disorder or motion. Glu127 and His89 are located nearby and each is hydrogen bonded to water molecules in the coordination sphere. Asp86 is not chelated to the magnesium or its surrounding water molecules. Results of kinetics and site-specific mutagenesis experiments suggest that this metal-water cluster contains the catalytic metal ion of this enzyme. All residues which hydrogen bond to the water molecules that coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved feature of this family of enzymes. We offer a detailed structural comparison to one other nuclease, the homing endonuclease I-PpoI, that has recently been shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these structures suggests that the magnesium of Serratia nuclease participates in catalysis via an inner sphere mechanism.     

Isolation of developmentally regulated genes in Drosophila melanogaster

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.     

Multiple catalase genes are differentially regulated in Aspergillus nidulans

Detoxification of hydrogen peroxide is a fundamental aspect of the cellular antioxidant responses in which catalases play a major role. Two differentially regulated catalase genes, catA and catB, have been studied in Aspergillus nidulans. Here we have characterized a third catalase gene, designated catC, which predicts a 475-amino-acid polypeptide containing a peroxisome-targeting signal. With a molecular mass of 54 kDa, CatC shows high similarity to other small-subunit monofunctional catalases and is most closely related to catalases from other fungi, Archaea, and animals. In contrast, the CatA (approximately 84 kDa) and CatB (approximately 79 kDa) enzymes belong to a family of large-subunit catalases, constituting a unique fungal and bacterial group. The catC gene displayed a relatively constant pattern of expression, not being induced by oxidative or other types of stress. Targeted disruption of catC eliminated a constitutive catalase activity not detected previously in zymogram gels. However, a catalase activity detected in catA catB mutant strains during late stationary phase was still present in catC and catABC null mutants, thus demonstrating the presence of a fourth catalase, here named catalase D (CatD). Neither catC nor catABC triple mutants showed any developmental defect, and both mutants grew as well as wild-type strains in H(2)O(2)-generating substrates, such as fatty acids, and/or purines as the sole carbon and nitrogen sources, respectively. CatD activity was induced during late stationary phase by glucose starvation, high temperature, and, to a lesser extent, H(2)O(2) treatment. The existence of at least four differentially regulated catalases indicates a large and regulated capability for H(2)O(2) detoxification in filamentous fungi.     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci.     

Salmonella typhi contains identical intervening sequences in all seven rrl genes.

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.     

Mouse ribosomal RNA genes contain multiple differentially regulated variants

Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.     

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis

Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is composed of two subunits of different molecular weights. Cytosolic GAPDH activity and protein both decreased immediately after transfer of 48-h rice seedlings to anaerobic conditions. Subsequent increase in activity and protein was accompanied by a change in isoenzyme profile and was preceded by an increase in steady-state messenger levels. One and two-dimensional electrophoretic analyses of in vivo and in vitro labeled GAPDH suggested that the change in isoenzyme profile under anaerobic conditions is due to preferential synthesis of one of the two GAPDH subunits caused by a specific increase in its mRNA.