Locations of three repetitive sequence families found in BALB/c adult beta-globin clones.

Three different repeat sequences have been mapped within the cloned EcoRI fragments that contain the adult beta-globin genes from the BALB/c (Hddd) mouse. One sequence, "a", occurs 1.5-2 kb 3' to the beta-major gene. A second, "b", is found 4kb 5' and 7.5kb 3' to the beta-minor gene. The 14kb EcoRI fragment bearing the beta-minor gene carries at least one additional repetitive element, "c". Probing a BALB/c DNA library with each repeat has demonstrated that these sequences are moderately to highly repetitive and are extensively interspersed with each other throughout the genome. In addition, repeats "a" and "b" are preferentially found in satellite and main-band DNa, respectively. The occurrence of these repeats elsewhere in the beta-globin cluster was demonstrated by probing the non-adult globin clones with each repeat. The arrangement of these repeats around the non-adult genes is 5'-"b"-"b"-epsilon y-beta hl-beta h2-"c"-beta h3-3'. Probing the C57BL/10 (Hbbs) adult gene clones with these repeats demonstrated that the distribution of these sequences in the adult region of these two haplotypes is essentially the same.     

The L1Md long interspersed repeat family in the mouse: almost all examples are truncated at one end.

We have characterized a large repetitive element which has been found at seven different locations within the beta globin locus of the BALB/c mouse. This repeat has an unusual structure in that each of the different members has the same end of the element conserved while the other end terminates at a different point in each repeat member. The sequences within the repeats from the beta globin locus have homology with other repetitive families such as the MIF-1, Bam-5, R, and the BamH1 families. These were recently proposed (T. Fanning, (1983) Nucleic Acids Res. 11, 5073-5091) to be part of a structure with the same organization which we found in the globin locus. Probing plaques from a BALB/c genomic library with sequences derived from the repeats in the globin locus shows that virtually all of the repeats from this family are organized in a manner consistent with the proposed structure.     

Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308.

The effect of a number of conditions on the amount of cyanophycin granule polypeptide [multi-L-arginyl poly(L-aspartic acid)] formed in the unicellular cyanobacterium Aphanocapsa 6308 was determined. Light, CO2, sulfur, and phosphorus starvation as well as the addition of arginine to culture media increased the amount of cyanophycin granule polypeptide in cells when compared with that in cells grown under conditions optimal for growth. Nitrogen limitation and reduction of growth temperature to 30 degrees C decreased the amount of cyanophycin granule polypeptide on a dry-weight basis. Shift-up and shift-down experiments suggest cyanophycin granule polypeptide may be a reserve nitrogen polymer in Aphanocapsa 6308.     

Nature and fate of the factors released during early contact interactions between hamster sperm and egg prior to fertilization in vitro

Initial attempts have been made to characterize three factors (Hartmann and Hutchison, 1977, J. Cell Physiol., 93, 41) which are released in vitro at 2, 31, and 50 min after capacitated hamster sperm make contact with but prior to penetration of the zona pellucida. A fourth factor is known to be released at 20–25 min but in the experiments described no effort was made toward its characterization. The assay for the factors is based upon their ability to induce early binding between gametes. Because the release of each of the factors occurs at a different time they can be harvested by sampling the supernatant at the appropriate time. The short-lived activity of these factors, which normally disappears soon after their release, was stabilized by buffering the medium to pH 7.0–7.4 with Tris or TES and removing the cells. Under these conditions, the factors released at 31 and 50 min were stable when incubated at 37°C in the absence of cells for at least 60 min, but the activity of the factor released at 2 min was erratic under similar incubation conditions. The factors released at 31 and 50 min passed unimpeded through filters with molecular weight cutoffs of 2000, and both eluted off Bio-Gel P-2 columns as single peaks of activity in regions corresponding to molecular weights of at least 1800 (void volume) and approximately 1400, respectively. The 2-min factor passed unimpeded through a filter capable of excluding molecules of molecular weight larger than 5000, but 50–86% of the activity was recovered after passing through a filter with a molecular weight cutoff at 2000. The release of the 2- and 31-min factors was inhibited 48 and 74%, respectively, by macromolecular trypsin inhibitors at concentrations which also blocked penetration of the egg by the sperm; these inhibitors had little or no effect on the release of the 50-min factor. The activity of ultrafiltered and pH-stabilized 31- and 50-min factor was destroyed by the proteases subtilisin and leucine aminopeptidase but was unaffected by trypsin or glycosidases. The disappearance of the 31- and 50- min factors after their release was investigated by incubating each factor with each of the cell types present in the drop and it was found that activity only declined in the presence of the eggs. Taken together, these results provide evidence that (1) at least two populations of peptides are released in a time-dependent manner when capacitated sperm make contact with the zona pellucida and (2) these peptides may disappear from the supernatant through an egg-mediated mechanism.     

The recognition site of type II restriction enzyme BglI is interrupted

The Type II restriction endonuclease BglI recognizes the interrupted DNA sequence 5'-G-C-C-N-N-N-N-N-G-G-C-. This sequence occurs at all locations in over 33 000 base pairs of DNA sequence where the enzyme was found to cut DNA and nowhere else. All six of the specified bases are essential parts of the site since all groups of five of the six bases occur in the DNA sequences tested and none of them are cut by BglI. The length of the block of intervening unspecified positions must be exactly five since all other sizes between zero and 15 occur in the DNA sequences searched and none are cut by BglI. The 5'-terminal nucleotides of BglI cleaved phage G4 replicative form DNA and plasmid pER18 DNA were compared with the DNA sequences near the BglI sites on these DNAs. These results indicated that BglI cuts within the intervening unspecified region and produces single-stranded 3' termini that are three bases long. The BglI recognition site and cleavage points can thus be represented as follows: (Formula: see text). This study of the BglI recognition site was facilitated by the use of inexpensive microcomputers. A system of programs was developed that allowed analysis of over 33 kb of DNA sequences stored on flexible magnetic disks or audio cassettes. While these programs were generally written in the higher level language BASIC, some assembly language subroutines were utilized to reduce execution time.     

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif.     

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.     

Hormonal control of behaviour: steroid action in the brain

There have recently been significant advances in our understanding of the cellular action of steroids on brain mechanisms of behaviour. Brain cells contain steroid metabolizing enzymes whose activity is modified by environmental stimuli. Steroids have rapid effects on neurotransmitter receptors via cell membranes and modify the distribution of neuropeptide receptors in areas controlling behaviour. It has been known for some time that oestrogens have an effect on brain structure that can be related to behaviour in the sexually dimorphic avian song system. Recent work suggests that oestrogen may have a similar effect on the developing sexually dimorphic nuclei of the mammalian brain.