Linkage of familial Hibernian fever to chromosome 12p13.

Autosomal dominant periodic fevers are characterized by intermittent febrile attacks of unknown etiology and by recurrent abdominal pains. The biochemical and molecular bases of all autosomal dominant periodic fevers are unknown, and only familial Hibernian fever (FHF) has been described as a distinct clinical entity. FHF has been reported in three families-the original Irish-Scottish family and two Irish families with similar clinical features. We have undertaken a genomewide search in these families and report significant multipoint LOD scores between the disease and markers on chromosome 12p13. Cumulative multipoint linkage analyses indicate that an FHF gene is likely to be located in an 8-cM interval between D12S77 and D12S356, with a maximum LOD score (Z max) of 3.79. The two-point Z max was 3.11, for D12S77. There was no evidence of genetic heterogeneity in these three families; it is proposed that these markers should be tested in other families, of different background, that have autosomal dominant periodic fever, as a prelude to identification of the FHF-susceptibility gene.     

Mouse beta defensin-1 is a functional homolog of human beta defensin-1

Defensin are 3–4 kDa antimicrobial peptides of which three distinct families have been identified; α-defensin, β-defensins, and insect defensins. Recent investigations have shown that β-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse β-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia. Received: 3 December 1997 / Accepted: 17 February 1998     

Fin duplications and deletions induced by disruption of retinoic acid signaling

 Retinoic acid (RA), a derivative of vitamin A, plays a critical role as a signaling molecule in axial patterning of vertebrates. Here we report that RA exposure of zebrafish (Danio rerio) and mummichog (Fundulus heteroclitus) embryos during gastrulation results in homeotic duplications of the pectoral fins in up to 94% of fish. We have observed three to four pairs of fins in an individual fish. Although some duplications are partial, many represent complete axial duplications of the pectoral girdle and fin and include coracoscapulae, proximal radials, and dermal fin elements. Fin duplications are observed only at a defined dose of RA. Inhibition of RA synthesis by exposure to citral during a narrow developmental window leads to fish which lack pectoral fins but can be rescued by addition of exogenous RA, suggesting that RA signaling is critical to fin specification during early development. The ability to consistently induce multiple fins in a large number of vertebrate embryos should contribute to the understanding of genetic regulation of the normal positioning of limbs during embryogenesis. Received: 30 August 1997 / Accepted: 6 December 1997     

The effect of pressure on the specific resistance of yeast filter cakes during dead-end filtration in the range 30–500kPa

Suspensions of Kluveromyces marxianus var. marxianus NRRLy2415 and active dry bakers' yeast were dead-end filtered in the range 30–500kPa. In all cases, the specific cake resistance, , could be related to pressure, P, through the expression = 0 (1 kcP) where 0 and kc are empirical constants. For K. marxianus, the values of kc were 1.67 × 10–5 Pa–1 and 2.39 × 10×5 Pa–1 for suspensions with mean cell aspect ratios of 2.98 and 7.33 respectively. Values of kcfor active dry yeast were 10.56 × 10–5 Pa–1 in the case of unwashed cells with a mean aspect ratio of 1.21 and 7.94 × 10–5 Pa–1 for washed cells with a mean aspect ratio of 1.20. © Rapid Science Ltd. 1998     

Analysis of 6 VNTR loci by ‘multiplex’ PCR and automated fluorescent detection

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to allelic dropout was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.     

Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat–, and Saccharomyces cerevisiae MAT1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.The first three authors contributed equally to the work     

An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.

An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.     

Taping the patella medially: a new treatment for osteoarthritis of the knee joint?

OBJECTIVE--To test the hypothesis that medial taping of the patella reduces the symptoms of osteoarthritis of the knee when the patellofemoral joint is affected. DESIGN--Randomised, single blind, crossover trial of three different forms of taping of the knee joint. Each tape (medial, lateral, or neutral) was applied for four days, with three days of no treatment between tape positions. SUBJECTS--14 patients with established, symptomatic osteoarthritis of the knee and both clinical and radiographic evidence of patellofemoral compartment disease. MAIN OUTCOME MEASURES--Daily visual analogue scale ratings for pain; patients'' rating of change with each treatment; and tape preference. RESULTS--Medial taping of the patella was significantly better than the neutral or lateral taping for pain scores, symptom change, and patient preference. The medial tape resulted in a 25% reduction in knee pain. CONCLUSION--Patella taping is a simple, safe, cheap way of providing short term pain relief in patients with osteoarthritis of the patellofemoral joint.     

The multifunctional polypeptide chain of rabbit mammary fatty acid synthase contains a domain homologous with the acyl carrier protein of Escherichia coli

The phosphopantetheine thiol of rabbit mammary fatty acid synthase was specifically alkylated using chloro[14C]acetyl-CoA and a radioactive fragment generated by limited elastase digestion of the modified protein was purified by gel filtration. We have previously mapped this fragment to an internal location in the 250 000-Mr polypeptide adjacent to the thioesterase domain [Eur. J. Biochem. 130, 185-193 (1983)]. The purified fragment had apparent molecular weights of 23 000 by gel filtration and 10 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while amino acid analysis indicated a minimal molecular weight of 10 400. We have determined the amino acid sequence of the first 64 residues of the fragment. The phosphopantetheine moiety is esterified to a serine at residue 38 in the sequence. When the sequences of the rabbit acyl carrier fragment and the 8847-Mr acyl carrier protein of Escherichia coli are aligned, 17 out of 64 residues are identical. These results suggest that the limited proteolysis delineates an internal acyl carrier domain within the rabbit protein and provide the first clear evidence that multifunctional fatty acid synthases have arisen by fusion of ancestral monofunctional proteins.