The sequence of the chromosomal mouse β-globin major gene: Homologies in capping,splicing and poly(A) sites

We have determined the entire nucleotide sequence of a cloned β-globin^maj gene derived from the BALB/c mouse. This sequence is 1567 bases long and includes the 5′ cap region as well as the presumptive poly(A) addition site of β-globin mRNA. The sequence establishes the fact that the gene is encoded in three discontinuous segments of DNA interrupted by two intervening sequences and precisely locates each. The smaller intervening sequence, 116 bases long, occurs between Arg and Leu codons at codon positions 30 and 31. The larger intervening sequence of 646 bases also occurs between Arg and Leu codons, but at codon positions 104 and 105. There is striking homology between the borders of the two intervening sequences, but no extensive dyad symmetry. Furthermore, the DNA region that just precedes and overlaps the 5′ cap structure of the mRNA shows homology to corresponding regions in other eucaryotic genes including the late adenovirus promoter. The 3′ untranslated sequence is closely homologous to that of the rabbit β-globin mRNA. The sequence thus allows us to identify several noncoding regions of potential importance for the expression and processing of genetic information. It also provides a basis for future comparison with other sequenced genes and a defined substrate for the development of direct tests of gene function.     

Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and has been used extensively for inducing mutations. In Arabidopsis, two methods for the isolation of genes identified on the basis of mutant phenotypes--genomic subtraction and chromosome walking--either rely on or are greatly facilitated by the availability of these types of mutations. This article gives a detailed characterization of ionizing radiation-induced mutations in plants. The Arabidopsis genes encoding chalcone flavanone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) were cloned and found to correspond to two transparent testa loci. A CHI allele, generated by fast-neutron irradiation, consisted of an inversion within the gene. A 272-bp fragment from 38 centimorgans away on the same chromosome was transferred to one end of this inversion. A DFR allele, induced by x-irradiation, contained two deletions and an inversion of the 2.8-centimorgan intervening region. Sequence analysis of the break points in both mutants indicate that repair of radiation-induced damage involves mechanisms similar or identical to those that mediate the integration of foreign sequences into the genome. The chromosome rearrangements found in these mutants have important implications for the use of ionizing radiation-induced alleles in classical and molecular genetic experiments in plants.     

Reduction of embryotoxicity by protein in embryo culture media.

Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentration, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.     

Disturbance regimes as determinants of seed banks in coastal dune vegetation of the southeastern Cape

Soil-stored seed banks of grassland, fynbos and thicket, all growing on calcareous dunes and each subject to different disturbance regimes, were examined. Seed banks were determined from counts of germinants from 50 soil cores from each type. Aboveground estimates of plant species cover in 10 1-m² plots were used in determining vegetation/seed bank similarities. There was no evidence for seed bank densities to be markedly higher in the most frequently disturbed community (grassland -4273 seeds/m²) than the least disturbed community (thicket - 3417 seeds/m²). Highest similarity between seed bank and above-ground vegetation composition in terms of species and growth form/life-span classes was recorded for grassland (CC = 50%). Lowest similarity (CC = 13%) was found in the less frequently disturbed thicket where no seeds of climax trees were recorded in the seed bank. A fynbos community on a north-facing (warm, dry) slope had intermediate-sized seed banks (1683 seeds/m²) with intermediate vegetation/seed bank similarity (CC = 46%). However, on the south-facing slope, which has a large post-fire ephemeral herb component, seed banks were larger (4518 seeds/m²) but less similar to above-ground vegetation (CC = 39%o). Ordination (DCA) of vegetation data from the four communities was different from an ordination of their seed bank data. Fynbos shrub species were absent from seed banks of both grassland and thicket, even though secondary succession proceeds from grassland, through fynbos to thicket. Their seed banks appear less persistent than those of European heath or Californian chaparral shrubs.     

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.     

The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

Smith degradation of gum exudates from some prosopis species

The polysaccharide of P. hymantophora has been shown to be composed of (1→4)-linked galactopyranosyl, (1→3)-linked galactopyranosyl, (1→3)-linked galactopyranosyl 2- and 4-sulphate and 2,6-disulphate residues. The (1→3)- and (1→4)-linked units are present in approximately equal amounts. The polysaccharide of P. hieroglyphica has been shown to possess (1→4)-linked galactopyranosyl, (1→3)-linked galactopyranosyl, and (1→3)-linked galactopyranosyl 2- and 4-sulphate residues. The (1→3)- and (1→4)-linked units are present in a 4:1 ratio. Both polysaccharides contain small proportions of non-reducing xylosyl end-groups.