Start sites for bidirectional in vitro DNA replication inside the replication origin, oriC, of Escherichia coli.

In vitro replication of mini-chromosomes in the absence of DNA ligase activity resulted in replication products with single-strand breaks at specific sites. The occurrence of these nicks was coupled to an active replication process, therefore we expect them to represent start sites for DNA replication. Two positions within oriC for each of the two leading strands of bidirectional replication were found. Within each position are one or two start sites. Counterclockwise synthesis started at positions 194/199 and 265/272, clockwise synthesis at positions 209/219 and 254. The start positions are located close to DnaA protein binding sites. A model for initiation accommodating this observation is discussed.     

Orientation and molecular map position of the complement genes in the mouse MHC.

Over the past few years six gene clusters have been isolated from the major histocompatibility complex (MHC) of the BALB/c mouse encompassing a total of 1600 kb of DNA and 48 genes. The molecular distances between these gene clusters and the orientation of four of the six clusters on chromosome 17 is not known. Here we use pulse-field gradient gels and Southern blot hybridization to establish large-scale genomic restriction maps covering several hundreds of kb surrounding the three gene clusters located in the K, I, S, and D regions of the MHC. Comparison of the maps orients the complement gene clusters in the S region with the 21-OHB gene pointing towards the K end and the C2 gene pointing towards the D end of the MHC. The distances between the E alpha and 21-OHB genes is 430 kb and between the C2 and TNF-alpha genes at least 420 kb.     

Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nucleotide changes which are exclusive to the chromosome 21 subfamily of human alphoid DNA. Southern blot analysis of alphoid repeat DNA on the chimpanzee X chromosome suggests this subfamily has been strongly conserved during and since the separation of chimpanzee and man although the two subfamilies can be distinguished on the basis of Taq I restriction fragments.     

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.     

Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP.

We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria. The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase. In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA. These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence. It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP. The incomplete chains were also more sensitive to proteinase K than the completed precursor. We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP. This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.     

Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin.

Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium. This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E. coli directly into the medium. We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E. coli outer membrane porin OmpF lacking its own N-terminal signal sequence. We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus. In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.     

Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential recombination between GC clusters in yeast mitochondrial DNA.

Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.