Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis.

Suppression subtractive hybridization, a cost-effective approach for targeting unique DNA, was used to identify a 41.7-kb Yersinia pestis-specific region. One primer pair designed from this region amplified PCR products from natural isolates of Y. pestis and produced no false positives for near neighbors, an important criterion for unambiguous bacterial identification.

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA.

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.

Vertical diplopia following local anaesthetic cataract surgery: predominantly a left eye problem?

PURPOSE: Vertical diplopia is an uncommon but disappointing complication of otherwise successful local anaesthetic cataract surgery. We studied strabismus patterns in a group of such patients to identify the nature and extent of extraocular muscle involvement. METHODS: A retrospective review identified 15 cases of vertical diplopia following local anaesthetic cataract surgery between July 1994 and January 1998. Peribulbar anaesthesia was used in all cases and given by right-handed professionals. RESULTS: All cases had otherwise successful cataract surgery (mean age 80.5 years; median pre-operative VA 6/18; median post-operative VA 6/9). The mean level of vertical diplopia was 7.2 prism dioptres (PD) in the primary position (range 2-25 PD). The left inferior rectus (IR) was paretic in 6 cases and restricted in 5 cases. The left superior rectus (SR) was not affected in any of the cases. The right IR was restricted in a single case. The right SR was paretic in 2 cases and restricted in a single case. None of the cases had clinical involvement of the oblique muscles. Eleven of the cases were managed successfully with prisms. Two of the cases required strabismus surgery. CONCLUSIONS: The incidence of left eye extraocular muscle involvement was greater than right eye involvement, although this did not reach statistical significance (73% vs 27%; p = 0.075). This may be due to the more difficult access of right-handed individuals giving left eye peribulbar injections with the needle tract being directed more closely to the muscle cone. The IR muscle is more commonly affected than the SR (80% vs 20%; p = 0.019). An equal incidence of paretic and restricted rectus muscle pathology was found in this study (53% vs 47%; p = 0.818). The exact aetiology of muscle injury is unknown but could be due to direct muscle or nerve trauma, anaesthetic toxicity, periocular haemorrhage or a combination of these.

Structure of the gene for congenital nephrotic syndrome of the finnish type (NPHS1) and characterization of mutations.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disorder that is caused by mutations in the recently discovered nephrin gene, NPHS1 (AF035835). The disease, which belongs to the Finnish disease heritage, exists predominantly in Finland, but many cases have been observed elsewhere in Europe and North America. The nephrin gene consists of 29 exons spanning 26 kb in the chromosomal region 19q13.1. In the present study, the genomic structure of the nephrin gene was analyzed, and 35 NPHS1 patients were screened for the presence of mutations in the gene. A total of 32 novel mutations, including deletions; insertions; nonsense, missense, and splicing mutations; and two common polymorphisms were found. Only two Swedish and four Finnish patients had the typical Finnish mutations: a 2-bp deletion in exon 2 (Finmajor) or a nonsense mutation in exon 26 (Finminor). In seven cases, no mutations were found in the coding region of the NPHS1 gene or in the immediate 5'-flanking region. These patients may have mutations elsewhere in the promoter, in intron areas, or in a gene encoding another protein that interacts with nephrin.

Structural organization of virulence-associated plasmids of Yersinia pestis.

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.

Complex beta-satellite repeat structures and the expansion of the zinc finger gene cluster in 19p12.

We investigated the organization, architecture, and evolution of the largest cluster ( approximately 4 Mb) of Krüppel-associated box zinc finger (KRAB-ZNF) genes located in cytogenetic band interval 19p12. A highly integrated physical map ( approximately 700 kb) of overlapping cosmid and BAC clones was developed between genetic STS markers D19S454 and D19S269. Using ZNF91 exon-specific probes to interrogate a detailed EcoRI restriction map of the region, ZNF genes were found to be distributed in a head-to-tail fashion throughout the region with an average density of one ZNF duplicon every 150-180 kb of genomic distance. Sequence analysis of 208,967 bp of this region indicated the presence of two putative ZNF genes: one consisting of a novel member of this gene family (ZNF208) expressed ubiquitously in all tissues examined and the other representing a nonprocessed pseudogene (ZNF209), located 450 kb proximal to ZNF208. Large blocks of ( approximately 25-kb) inverted beta-satellite repeats with a remarkably symmetrical higher order repeat structure were found to bracket the functional ZNF gene. Hybridization analysis using the beta-satellite repeat as a probe indicates that beta-satellite interspersion between ZNF gene cassettes is a general property for 1.5 Mb of the ZNF gene cluster in 19p12. Both molecular clock data as well as a retroposon-mapping molecular fossil approach indicate that this ZNF cluster arose early during primate evolution (approximately 50 million years ago). We propose an evolutionary model in which heteromorphic pericentromeric repeat structures such as the beta satellites have been coopted to accommodate rapid expansion of a large gene family over a short period of evolutionary time. [The sequence data described in this paper have been submitted to GenBank under accession nos. AC003973 and AC004004.]

Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.

Structure of the human amyloid-precursor-like protein gene APLP1 at 19q13.1.

Amyloid-precursor-like protein 1 (APLP1) is a membrane-associated glycoprotein, whose gene is homologous to the APP gene, which has been shown to be involved in the pathogenesis of Alzheimer's disease. APLP1 is predominantly expressed in brain, particularly in the cerebral cortex postsynaptic density. The genomic organization of mouse APLP1 has been determined, and the human gene has been mapped to chromosomal region 19q13.1. In the present study, the entire sequence of human APLP1 has been determined from a cosmid clone, and the genomic structure has been determined. The gene is 11.8 kb long and contains 17 exons. We have previously mapped the gene for congenital nephrotic syndrome (CNF) to the APLP1 region, to the vicinity of marker D19S610 located between markers D19S191 and DS19608. APLP1 is the only known gene in the vicinity of the marker D19S610. Because of its location and the proposed interference of amyloid with basement membrane assembly, APLP1 has been considered a candidate gene for CNF. All exon regions of the gene were amplified by the polymerase chain reaction and sequenced from DNA of CNF patients. No differences were observed between CNF patients and controls, suggesting that mutations in APLP1 are not involved in the etiology of CNF.

Multiple negative and positive cis-acting elements control the expression of the murine CD4 gene.

The cis-acting elements located within 15 kb 5' of the murine CD4 gene transcriptional start site and the first intron of the CD4 gene have been investigated using deletion constructs. Our transient transfection data indicate that the expression of the murine CD4 gene is controlled by multiple positive and negative regulatory cis-acting elements. There are at least two cis-acting elements that have a positive effect on the expression of the CD4 gene and at least four regions of DNA that have a negative effect. The positive control elements are located about 13.5 kb 5' of the promoter and within the flanking sequences of the first intron. The DNA between the 5' enhancer and the promoter contains at least two regions that exert a negative effect on CD4 expression. In addition to the positive effect that the first intron has on CD4 expression, there are two regions within the first intron that have a negative effect. These two negative regulatory elements correspond to two T-cell-specific DNase I hypersensitive sites found in the first intron.

Regulation of Bacillus subtilis senS by homologous regulatory regions of senS and the inducible cat gene.

We have identified a stem-and-loop region between the promoter and open reading frame of the Bacillus subtilis senS gene containing a 9 base sequence (TTTAGATAA) identical to a sequence found in a similar regulatory element of the inducible pC194 cat gene. A high copy number of this pC194 regulatory region repressed the expression of senS, indicating that the stem-and-loop region of the cat gene titrated a positive factor necessary for senS expression. These results suggest that factors with similar binding requirements control senS and cat expression. In addition, senS was found to be autoregulated.

Bacillus subtilis SenS exerts its activity through a site in the 5' flanking region of the aprE promoter.

The Bacillus subtilis gene senS, when present in high copy number, stimulates the expression of several extracellular protein genes during the onset of stationary phase, e.g. aprE. A novel integration vector, pINT, was constructed for transcription expression studies; it employed a unique method of promoter insert production for fusion with the lacZ reporter gene. Deletions were made of the 5' flanking region of the aprE promoter to localize the site responsible for SenS-mediated enhancement activity. pINT was used translationally fuse aprE promoter deletion fragments with the lacZ reporter gene. A site between -177 and -415 with respect to the aprE start site of transcription was found to be required for the maximal SenS-mediated transcription increase from the aprE promoter. A multicopy vector containing the senS coding region without its native negative regulation was highly unstable in B. subtilis; this was due to the expressed senS insert.

Detection of simian immunodeficiency virus RNA from infected rhesus macaques by the polymerase chain reaction.

A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIVmac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 x 10(5) PBMC of two animals.

Portable system for the measurement of heart rate.

We describe a simple RR interval recording system; it does not involve an analogue recording of ECG data on magnetic tape and does not therefore suffer from the inherent problem of tape speed variability on playback, which is a frequent cause of inaccuracies in RR interval timing. The system operates in both recording and playback modes and records of up to 60 minutes duration can be obtained without operator intervention. Its accuracy with respect to RR interval is not less than 99.5%.

Audit of a general practice.

During a period of one full week, an analysis was made of the activities of patients (demand) and workers (service) in a suburban general practice. A data collection system was constructed and used by all the health centre workers. We sought to measure demand and service and to show the value of measurement (audit) of an aspect of service, i.e. prescribing.

The topography of the superificial roots and ganglia of the anterior lateral line nerve of the smooth dogfish, Mustelus canis.

The topographic relationships of the superficial roots and ganglia of the anterior lateral line nerve (NLLa) to each other and to cranial nerves V, VII and VIII were studied, in the smooth dogfish Mustelus canis, by dissection of Sudan Black B stained specimens. NLLa consists of four branches: namely, superficial ophthalmic, buccal, otic and external mandibular. Each branch carriers lateral line neurons exclusively and forms a dorsal root and a ventral root which enter the anterior lateral line lobe and posterior lateral line lobe of the medulla respectively. It is estimated that slightly more than 50% of the fibers of the superficial ophthalmic, approximately 60% of the fibers of the buccal, at least 35% of the fibers of the otic and about 20% of the fibers of the external mandibular constitute the dorsal root. The ventral root is comprised of less than 50% of the fibers of the superficial ophthalmic, 40% of the fibers of the buccal, at least 50% of the fibers of the otic, and 80% of the fibers of the external mandibular. These results are correlated with the peripheral distribution and central termination of NLLa and it is concluded that the dorsal root carries axons from ampullary receptors and the ventral root carries fibers that innervate ordinary lateral line sense organs.