Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites.

The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition.     

Absence of Turner stigmata in a 46,XYp-female

A 46,XY female patient with streak gonads and a large deletion of Yp is described. The deletion included the Y chromosomal genes SRY, ZFY, and RPS4Y. The patient did not display any Turner stigmata, such as webbing of the neck, cardiac or other abnormalities. The findings argue against an important role of RPS4Y in the prevention of Turner stigmata in males and are consistent with a role of SRY in testis differentiation in humans.     

The Machado-Joseph disease locus is different from the spinocerebellar ataxia locus (SCA1).

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia that has been described primarily in families of Azorean or Portuguese descent. MJD and chromosome 6p-linked spinocerebellar ataxia (SCA1) are difficult to differentiate clinically, and it has been suggested that they may be allelic variants of the same disorder. We have tested MJD families for linkage to six DNA sequence polymorphisms located on chromosome 6p, including the highly informative dinucleotide repeat, D6S89. Seventeen centimorgans telomeric to and 41 cM centromeric to D6S89, a region that includes the SCA1 locus reported to be within 3 cM of D6S89, have been excluded. These data provide conclusive evidence that MJD and SCA1 are nonallelic.     

Cyclic behavior of plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), within caged dwarf apple trees in spring

From 12 to 19 May 1987, during Morspur apple bloom, 21 radioactively labeled (⁶⁵ Zn) adult plum curculios, Conotrachelus nenuphar (Herbst), were released within a field cage containing four dwarf apple trees and located three times a day. A technique was developed for quickly obtaining (x, y, z)coordinates of location for adults foraging within apple trees. Cyclic patterns of behavior were detected using spectral analysis procedures. Over 70% of plum curculios exhibited diel periodicity with respect to activity and rate of movement, 36% exhibited such periodicity with respect to presence in the trees, and 27% with respect to movements from the center to the periphery of the canopy. Presence in fruit clusters, height in the trees, and movements along east-west and north-south axes showed little or no periodicity. Factors triggering cyclic behavior and practical implications of the results are discussed.     

Genomic targets of the serendipity beta and delta zinc finger proteins and their respective DNA recognition sites.

The closely related Drosophila serendipity (sry) beta (beta) and delta (delta) Cys2-His2 zinc finger proteins show partly overlapping in vitro DNA binding specificities and distinct patterns of binding sites on polytene chromosomes. Using a newly developed procedure, we identified genomic DNA targets for these two proteins. Both the sry beta and delta proteins protect an 18-22 base region from DNase I digestion within each analysed genomic binding site, that includes a 13 bp consensus sequence. The consensus recognition sites sry beta 5'-YCAGAGATGCGCA-3' and sry delta 5'-YTAGAGATGGRAA-3' thus differ by nucleotides at four out of 13 positions. They are determinant for specific binding of the sry beta and delta proteins, respectively, produced in Escherichia coli or present in Drosophila embryos. We further show that sry beta is the major (if not exclusive) Drosophila nuclear protein that specifically binds the 5'-CAGAGTGCGC-3' sequence. The identified sry beta genomic targets are all contained within single-copy DNA in euchromatic regions of the genome. Two out of the five characterized in detail map at cytological positions coincident with binding sites of the native sry beta protein on polytene chromosomes.     

Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families.

We have characterized and genetically mapped new polymorphic DNA markers in the q27-q28 region of the X chromosome. New informative RFLPs have been found for DXS105, DXS115, and DXS152. In particular, heterozygosity at the DXS105 locus has been increased from 25% to 52%. We have shown that DXS105 and DXS152 are contained within a 40-kb region. A multipoint linkage analysis was performed in fragile-X families and in large normal families from the Centre d'Etudes du Polymorphisme Humain (CEPH). This has allowed us to establish the order centromere-DXS144-DXS51-DXS102-F9-DXS105-FRAX A-(F8, DXS15, DXS52, DXS115). DXS102 is close to the hemophilia-B locus (z[theta] = 13.6 at theta = .02) and might thus be used as an alternative probe for diagnosis in Hemophila-B families not informative for intragenic RFLPs. DXS105 is 8% recombination closer to the fragile-X locus than F9 (z[theta] = 14.6 at theta = .08 for the F9-DXS105 linkage) and should thus be a better marker for analysis of fragile-X families. However, the DXS105 locus appears to be still loosely linked to the fragile-X locus in some families. The multipoint estimation for recombination between DXS105 and FRAXA is .16 in our set of data. Our data indicate that the region responsible for the heterogeneity in recombination between F9 and the fragile-X locus is within the DXS105-FRAXA interval.