Detection of repeat expansions in large next generation DNA and RNA sequencing data without alignment.

Bioinformatic methods for detecting short tandem repeat expansions in short-read sequencing have identified new repeat expansions in humans, but require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment. superSTR is used to process whole-genome and whole-exome sequencing data, and perform the first STR analysis of the UK Biobank, efficiently screening and identifying known and potential disease-associated STRs in the exomes of 49,953 biobank participants. We demonstrate the first bioinformatic screening of RNA sequencing data to detect repeat expansions in humans and mouse models of ataxia and dystrophy.

Is there a morning-evening difference in egg production of bloodworms, strongyle parasites of equines?

To test a claim that counts of the eggs of large strongyles or bloodworms, parasites of equines, are greater in morning fecal samples than in those from later in the day, and therefore, may be indicators of circadian variations in these worms, samples from one pony and nine horses were analyzed for strongyle eggs. Samples were collected twice per day, about 7:30 and again about 19:30, during two, 24-day periods. Individual counts varied from 0 to 1184. The mean of all 7:30 counts was 212.52; that of 19:30 counts was 236.03. The difference is not significant, and does not support the idea of circadian differences in egg production of these nematodes.

Stress and changes in the blood of newts, Notophthalmus viridescens, during early regeneration.

During the summers of 1984 and 1985, adult red-spotted newts, Notophthalmus viridescens, were maintained in the laboratory at 23 degrees +/- 0.5 degrees C under natural photoperiods. From each of the experimental animals, the right forelimb was amputated just proximal to the elbow. Control newts were not manipulated surgically. Eight, 15, and 22 days after the time of amputation, equal numbers of regenerating and control animals were sacrificed, and blood smears of each individual were prepared with Wright's stain. Mean differential counts of leukocytes of the two groups of newts indicated that the relative number of neutrophils increased and the relative number of lymphocytes decreased in the regenerating animals as compared to their controls (Fig. 1 and Fig. 2). Earlier studies had shown that lymphopenia and neutrophilia occur in red-spotted newts treated with hydrocortisone or with ACTH or subjected to environmental stress (Bennett and Daigle 1983). Consequently, it is suggested that amputation and/or early regeneration may stimulate the increased production of hormones associated with stress in vertebrates, which may, in turn, influence regeneration, itself, and that the detailed study of the distribution of leukocytes in Notophthalmus viridescens may provide an assay with which to study the regulation of regeneration in this species.

Identification in Xenopus of a structural homologue of the Drosophila gene snail.

We have cloned a Xenopus cDNA that is related to snail, a gene that is required for mesoderm formation in Drosophila. The cDNA encodes a protein that contains five zinc-fingers that closely resemble those of snail. In the non-canonical parts of the DNA-binding loop, there is almost 90% homology between snail and xsna. The corresponding mRNA (xsna) is expressed strongly at the start of zygotic transcription simultaneously with the transcription factor EF1 alpha. In early gastrulae, xsna is equally distributed between the dorsal and ventral halves of the equatorial zone. The possibility that the capacity to synthesise xsna is more localised before the start of zygotic transcription has been investigated by culturing fragments of stage 8 embryos until xsna is synthesised. The capacity to synthesise xsna at stage 8 is located principally in the dorsal half of the equatorial zone. A small amount of maternal xsna is localised in the vegetal hemisphere before zygotic transcription starts. xsna is not present in isolated animal caps but can be induced by the mesoderm-inducing factors XTC-MIF and bFGF. Synthesis of xsna does not occur autonomously in dispersed cells but is restored when cells reaggregate in the presence of calcium and magnesium.

Amplification of a major membrane-bound DNA sequence of Bacillus subtilis.

A membrane-bound DNA sequence from Bacillus subtilis was subcloned into a plasmid which can replicate in Escherichia coli but not in B. subtilis. This plasmid hybridized with an 11-kilobase HindIII fragment which is the major particle-bound fragment in lysates treated with HindIII. The plasmid integrated into the B. subtilis chromosome at the region of homology, conferring chloramphenicol resistance on the recipient. The inserted resistance was mapped close to purA by using the generalized transducing phage AR9. In one chloramphenicol-resistant strain, the pMS31 region was repeated at least 20 times. A large proportion of the copies of the cloned region were present in the particle fraction, indicating that the capacity to bind this region of the chromosome was substantially in excess of the normal dose of the region. The structure of the particle-bound region was sensitive to ionic detergents and high salt concentrations but was not greatly affected by RNase or ethidium bromide. The basis of a specific DNA-membrane interaction can now be studied by using the amplified region, without the complications of sequences required for autonomous plasmid replication.

Identification of a specific membrane-particle-associated DNA sequence in Bacillus subtilis.

After the Bacillus subtilis nucleoid was dissected with restriction endonucleases, a specific DNA sequence from the purA region was isolated in a particulate form that probably originated from the cell membrane. Precise definition of the binding region within this sequence was achieved by a novel procedure based on a previously reported observation that additional copies of the binding region, introduced into the chromosome using an integrative plasmid, were also predominantly particle bound. Subsections of the original plasmid insertion were cloned into the integrative plasmid and introduced into B. subtilis, in which they became tandemly reiterated under appropriate selective conditions. HaeIII sites in the vector, flanking each insertion, were used to excise the latter for subsequent tests of particle association. Examination of 10 strains containing subsections of the original 5.2-kilobase-pair region showed that the binding region was confined to 283 base pairs. This was confirmed by dissection in vitro of a larger, isolated, particle-bound sequence. The nucleotide sequence of a 1,300-base-pair region that contained this site was determined. The entire region had a notably high A + T content and was deficient in open reading frames for transcription.

Attachment of the chromosomal terminus of Bacillus subtilis to a fast-sedimenting particle.

After gently lysed protoplasts of exponential phase cells of Bacillus subtilis were treated with restriction endonuclease BamHI, 99% of the DNA did not sediment with the plasma membrane. This DNA was fractionated on sucrose gradients into (i) a fast-sedimenting fraction highly enriched for genes from the origin and terminus (purA and ilvA), (ii) a 50 to 100S component also enriched for purA and ilvA, and (iii) the bulk of the DNA. The fast-sedimenting fraction was dissociated by Sarkosyl; this fraction contained a substantial amount of protein and is probably a membrane subparticle. The S value of the 50 to 100S component was not greatly affected by Sarkosyl treatment, but these particles were unable to penetrate an agarose gel during electrophoresis and were retained by nitrocellulose filters. The terminus DNA in the fast-sedimenting fraction and the 50 to 100S component contained a large restriction fragment (1.5 x 10(7) to 2.0 x 10(7) daltons) encoding ilvA, thyB, and ilvD. The bulk of the SP beta prophage and metB, which lie to the right and left, respectively, of the ilvA-ilvD cluster, were not part of the complex. citK, which lies to the right of SP beta, appeared to be present in the fast-sedimenting complexes. The neighboring genes kauA and gltA were not part of the fast-sedimenting complexes. The presence of terminus DNA in the fast-sedimenting components was also demonstrated by a radiochemical method.

Potentiation of a nucleolytic activity in Bacillus subtilis.

In several strains of Bacillus subtilis extensive breakdown of chromosomal DNA may be potentiated by osmotic lysis of protoplasts. At its most severe, in strains originating from Farmer & Rothman's thymine auxotroph, the rate of DNA breakdown was greater than 50% per hour at 40 degrees C. The rate of DNA breakdown in most other strains tested was approximately 5% per hour except for SP beta- strains, in which the rate of DNA breakdown was only 0.3%. DNA degradation was attributed to relaxation of control of a nuclease specified by the prophage of SP beta or a related phage. The most potent nuclease in lysates was an ATP-activated protein of Mr 280 000. Derivatives of Farmer and Rothman's strain containing integrated plasmids had the highest rate of DNA degradation. Although the chromosome was completely destroyed, covalently closed circular plasmids were generated from the integrated sequence. These showed massive deletions of the B. subtilis part of the integrated plasmid but the vector sequence remained intact. The nucleolytic activity therefore appears to recognize specific sequences in B. subtilis DNA. We suggest that activation of SP beta genes during development of competence may be a cause of deletion of cloned genes in the early stages of establishment of cloned sequences.

Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis.

When lysates of Bacillus subtilis were treated with restriction endonucleases EcoRI or HindIII, almost all of the DNA was released from the major plasma membrane fraction that was sedimentable at low speed. However, a very small part of the released DNA, when centrifuged at high speed, appeared to be bound to small membrane fragments. On agarose gels, this material, prepared with either enzyme, contained only a small number of restriction fragments, and the DNA in the sample hybridized with 11 to 12 EcoRI or HindIII fragments of chromosomal DNA. This DNA was used after nick-translation to screen Charon 4A clone banks for phages containing membrane-bound fragments. One of these was studied in detail. Only a part (about 5 kilobases) of the region present in this clone is important in binding the DNA to the membrane subparticle.

Distinct elements of the xsna promoter are required for mesodermal and ectodermal expression.

Xsna, the Xenopus homologue of Drosophila snail, is expressed in both mesoderm and ectoderm. Expression occurs in all mesoderm initially but is down regulated in a tissue-specific fashion at the end of gastrulation in a way that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated. Xsna is also expressed in the ectoderm of the prospective neural fold from stage 11, in a distinct band of cells surrounding the prospective neural plate, which we designate the neural plate border. The deep and superficial ectoderm compartments labelled by Xsna represent the prospective neural crest and the prospective roof of the neural tube, respectively. Xsna expression persists in neural crest cells during their subsequent migration. The role of the Xsna promoter in creating this pattern of expression has been investigated by injecting fertilised eggs with constructs containing the 5' upstream sequence of the gene fused to a reporter. An element of 115 base pairs (-160 to -45 relative to the transcriptional start) is sufficient to drive appropriate reporter gene expression. The promoter does not contain a TATA or CAAT box and does not have a high GC content, but RNA synthesis starts precisely at 33 bases upstream to the translational start. The start sequence can be deleted so that transcription is initiated elsewhere without affecting the expression pattern. The distribution of Xsna promoter activity within the embryo, examined using beta-galactosidase (beta-gal) fusions, is similar to that of the endogenous mRNA seen by in situ hybridisation. The contribution of elements within the 5' sequence have been assessed by comparing the expression patterns of constructs that have deletions in this region. Sequences from -112 to -97 are required for mesodermal expression and sequences from -96 to -44 are required for ectodermal expression. The behaviour of the injected promoter constructs differ in one important respect from the endogenous gene in that expression in an animal cap assay is not inducible by mesoderm-inducing factors but is inducible by cells of the vegetal pole.

Cloning and developmental expression of Sna, a murine homologue of the Drosophila snail gene.

The genetic analysis of dorsoventral patterning in Drosophila has identified a zinc-finger gene, snail, that is required for mesoderm formation. The cloning and nuclease protection analysis of a Xenopus homologue of this gene has suggested a possible role in the mesoderm of vertebrates. Here, we describe the cloning of a murine homologue of snail, Sna, and in situ hybridisation studies of its developmental expression. Sequence analysis reveals substantial conservation of the second to fifth zinc fingers, but not of the first zinc finger in the Sna gene. Expression occurs in the ectoplacental cone, parietal endoderm, embryonic and extraembryonic mesoderm, in neural crest and in condensing precartilage. Based on the timing and spatial restriction of expression in embryonic mesoderm, we suggest that Sna might be required for the early development of this tissue, as is the case for its Drosophila counterpart. In addition, we propose that Sna might have an analogous role in the development of neural crest. The expression in condensing precartilage indicates that this gene also has a later function in chondrogenesis.