Organization of simple sequences in the Drosophilia melanoga ter genome

Fragments of Drosophila melanogaster DNA that intensively hybridize with simple sequences poly[(dG-dT).(dC-dA)], poly[(dA).(dT)] and poly[(dG-dA).(dC-dT)] were cloned. The first two types of simple sequences are organized in these clones as separated stretches of moderate length, repeated many times within 12-15 kb. Each cluster contains only one type of the simple sequences and originates from a unique in the genome. In contrast, poly[(dG-dA).(dC-dT)] occurs in the genome as several isolated motifs.     

The capacity to form H-DNA cannot substitute for GAGA factor binding to a (CT)n*(GA)n regulatory site

Previous studies of the Drosophila melanogaster hsp26 gene promoter have demonstrated the importance of a homopurine·homopyrimidine segment [primarily (CT)_n·(GA)_n] for chromatin structure formation and gene activation. (CT)_n regions are known to bind GAGA factor, a dominant enhancer of PEV thought to play a role in generating an accessible chromatin structure. The (CT)_n region can also form an H-DNA structure in vitro under acidic pH and negative supercoiling; a detailed map of that structure is reported here. To test whether the (CT)_n sequence can function through H-DNA in vivo, we have analyzed a series of hsp26-lacZ transgenes with altered sequences in this region. The results indicate that a 25 bp mirror repeat within the homopurine·homopyrimidine region, while adequate for H-DNA formation, is neither necessary nor sufficient for positive regulation of hsp26 when GAGA factor-binding sites have been eliminated. The ability to form H-DNA cannot substitute for GAGA factor binding to the (CT)_n sequence.     

Nuclear proteins from Drosophila melanogaster embryos which specifically bind to simple homopolymeric sequences poly [(dT-dG).(dC-dA)]

The binding of nuclear proteins from Drosophila melanogaster embryos to simple homopolymeric DNA sequences was studied. Nuclear proteins were electrophoresed, transferred onto nitrocellulose and incubated with labelled synthetic homopolymers or natural fragment containing simple sequences. Several protein bands were found in the 65-72 KDa region, which specifically bind both poly [(dG-dT).(dA-dC)] and a natural fragment containing 40 bp of this sequence. These proteins do not bind to homopolymers poly [(dA).(dT)] and poly [(dG-dA).(dC-dT)], or other foreign DNAs.     

Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex

Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine-homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G-CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues.     

The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA.

Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.     

Hsp90-mediated assembly of the 26 S proteasome is involved in major histocompatibility complex class I antigen processing

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.     

Multiple, compensatory regulatory elements specify spermatocyte-specific expression of the Drosophila melanogaster hsp26 gene.

The hsp26 gene of Drosophila melanogaster is expressed in six tissues during development and in a tissue-general response to heat shock. To be able to compare tissue-specific and heat-induced mechanisms of hsp26 expression, we have begun an analysis of the sequences involved in the spermatocyte-specific expression of the hsp26 gene by using germ line transformation. hsp26 mRNA synthesized in the spermatocytes has the same start site as sites previously demonstrated for nurse cell-specific and heat-induced mRNAs. Three regions of the hsp26 gene (nucleotides -351 to -135, -135 to -85, and +11 to +632) were able to stimulate spermatocyte-specific expression when fused with promoter sequences (nucleotides -85 to +11) that alone were insufficient to stimulate expression. These stimulatory regions appear to contain elements that provide redundant functions. While each region was able to stimulate expression independently, the deletion of any one region from a construct was without consequence as long as another compensatory region(s) was still present. There must reside, at a minimum, two independent spermatocyte-specifying elements within the sequences that encompass the three stimulatory regions and the promoter. At least one element is contained within sequences from -351 to -48. This region, in either orientation, can stimulate spermatocyte-specific expression from a heterologous promoter. A second element must reside in sequences from -52 to +632, since these sequences are also sufficient to direct spermatocyte-specific expression.     

Short Communication Involvement of Calcium-calmodulin in the Expression of hsp26 Gene in Wheat

The treatment of wheat ( Triticum aestivum L.) seedlings with CaCl2 increased mRNA levels of hsp26 gene at 37 ℃ for 1 h, while that with Ca2+ chelator EGTA decreased the expression of the hsp26 gene. The expression of the wheat calmodulin gene CaM1-2 is rapidly up-regulated in wheat seedlings by heat shock at 37 ℃. The heat-induced up-regulation of CaM1-2 occurred earlier than that of the hsp26 gene did. Calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamid (W7) inhibited the expression of the hsp26 gene in wheat seedlings at 37 ℃ for 1 h. These results implied that the calcium-calmodulin is probably involved in the signal transduction of the hsp26 gene expression induced by heat shock.     

H-DNA formation by the coding repeat elements of neisserial opa genes

The coding repeat region of opa genes from Neisseria gonorrhoeae and Neisseria meningitidis determines the expression state of their respective genes through high-frequency addition of deletion of pentanucleotide coding repeat units (CRs; CTTCT). In vitro analyses of cloned opa gene CR regions using single-strand specific nucleases, oligonucleotide protection experiments, and modifications of non-B-DNA residues indicate that the regions form structures resembling H-DNA under acidic conditions in the presence of negative supercoiling. The purine/pyrimidine strand bias and H-palindromic nature of the repeat region are consistent with sequence requirements for H-DNA formation. Sequences flanking the repeat elements are required to form the H-DNA structure in vitro as judged by the pattern of exposed non-B-DNA residues in CR sequences synthesized as oligonucleotides to form beta-galactosidase::CR translational fusions. The fusions phase vary by addition and deletion of CR elements and the rate of phase variation increases upon induction of the fusion genes. The opa gene CR region is the first reported bacterial H-DNA structure and is unique in that it lies within the coding sequence for the gene.     

Simple repetitive sequences in the genomes of archaebacteria

Stretches of simple sequences poly(dG-dT).poly(dC-dA), poly(dG-dA).poly(dC-dT), poly(dG).poly(dC) and poly(dA).poly(dT), the occurrence of which is a characteristic feature of eukaryotic genomes, are found in the genomes of archaebacteria Halobacterium halobium and Sulfolobus acidocaldarius. In S. acidocaldarius these sequences constitute a considerable portion of the genome; they belong to a class of repetitive sequences dispersed throughout the genome, being transcribed and found in RNAs of different lengths.     

Upregulation of heat shock protein 32 in Sertoli cells alleviates the impairments caused by heat shock-induced apoptosis in mouse testis

Heat stress results in apoptosis in testicular germ cells. A small heat shock protein (hsp), hsp32, is induced by heat stress in the testis, but little is known about its definitive function in physiological processes. To clarify the underlying role of hsp32, hsp32 expression and related signals in the heat shock pathway were analysed in mouse testes and Sertoli cells after heat stress in vivo and in vitro; meanwhile, expression of hsp32 was silenced only in the Sertoli cells using three different small interfering RNAs (siRNAs) to further verify the functional role of hsp32 in Sertoli cells, and hsp32-derived carbon monoxide (CO) contents in cultured media were analysed to clarify whether hsp32-derived CO involve in the apoptosis regulation mechanisms. The results from the in vivo experiment showed that the high expression levels of hsp32 (P?<?0.05) were observed whether chronic, moderate or acute, transient heat exposure. The in vitro experiment showed that acute, transient heat exposure resulted in increases in Sertoli cells apoptosis (P?<?0.01), the expression of hsp32 and caspase-3 activity; hsp32-siRNA knockdown of hsp32 expression resulted in upregulated apoptosis (P?<?0.01) and caspase-3 activity (P?<?0.01) in the Sertoli cells and hyperthermia increases CO (P?<?0.01) release by Sertoli cells. The results suggested that upregulating hsp32 in Sertoli cells inhibits caspase-3 activity and alleviates heat-induced impairments in mouse testis; hsp32-derived CO may involve in the regulation mechanism.     

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.     

Structural polymorphism of homopurine--homopyrimidine sequences: the secondary DNA structure adopted by a d(GA.CT)22 sequence in the presence of zinc ions.

In this paper, we have analysed the conformational behaviour shown by the homopurine--homopyrimidine alternating d(GA.CT)22 sequence cloned into SV40. Our results show that, in the presence of zinc ions, the d(GA.CT)22 sequence adopts an altered secondary DNA structure (*H-DNA) which differs from either B-DNA or H-DNA. Formation of *H-DNA is facilitated by negative supercoiling and does not appear to require base protonation, since it is induced at neutral pH by approximately 0.4 mM ZnCl2. The patterns of OsO4 and DEPC modification obtained in the presence of zinc are compatible with a homopurine--homopurine--homopyridimine triplex, though other structural models for *H-DNA are also possible. The hypersensitivity to S1-cleavage of the d(GA.CT)22 sequence is reinterpreted in terms of the equilibria between the B-, H- and *H-forms of the sequence. These results reveal the high degree of structural polymorphism shown by homopurine-homopyrimidine sequences. Its biological relevance is discussed.