Characterization of a human ''midisatellite'' sequence.

We have examined the structure and DNA sequence of a human genomic locus that consists of a large hypervariable region made up of repeats of a simple sequence. With several restriction enzymes, the locus shows many restriction fragments that vary quantitatively as well as qualitatively. Other restriction enzymes produce only a single, high-molecular-weight fragment at this locus. Almost all of the fragments are revealed with a simple sequence probe. Southern transfers of the high-molecular-weight restriction fragments produced by the restriction enzymes NotI and SfiI, resolved by pulsed-field gel electrophoresis, gave at most two fragments, demonstrated to be allelic, showing that the majority of the restriction fragments seen in the complex patterns are at a single locus. The estimated size of the region homologous to the probe varied from 250 to 500 kilobases. DNA sequencing indicated that the region consists of tandem repeats of a 40-base-pair sequence. Some homology was detected to the tandem repeating units of the insulin gene and the zetaglobin pseudogene hypervariable regions, and to the "minisatellite" DNA at the myoglobin locus.     

cDNA cloning and sequencing of tarantula hemocyanin subunits

Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.     

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.     

Interleukin 2 receptors on cultured murine epidermal Langerhans cells

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Proteolysis of tubulin and the substructure of the tubulin dimer

The alpha and beta subunits of tubulin each have a single highly reactive site for a variety of proteases that divides each subunit into two unequal regions. The position of cleavage is not the same for alpha and beta, since alpha is consistently cleaved into about 38- and 14-kDa pieces, while beta is cleaved into about 34- and 21-kDa pieces. The larger fragment is amino-terminal in both subunits as shown: by size reduction of the smaller fragment by subtilisin (which cleaves at the extreme carboxyl-terminal end), but no change in size of the larger fragment; by the charge/mass ratios of the proteolytic fragments; and by sequence analysis which locates trypsin cleavage after residue 339 (alpha) and chymotrypsin cleavage after residue 281 (beta). Since this cleavage pattern of the alpha and beta subunits is found for very different proteases, we suggest that it is determined by structural features of the tubulin molecule. The two pieces of each subunit remain associated following cleavage. While both cleavage sites are exposed in the free dimer, assembly of dimers into microtubules or sheets protects the internal site against cleavage. By contrast, the carboxyl-terminal subtilisin-sensitive sites remain exposed. Based on these results we propose a model for the substructure of the tubulin dimer that accommodates internal cleavage in the dimer but not the polymer, access to the COOH termini in both forms, and the orientation of the dimer in the polymer.