A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K252a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.

Physical map and cosmid contig encompassing a new interstitial deletion of the X-linked lymphoproliferative syndrome region.

The X-linked lymphoproliferative syndrome (XLP) is an inherited immuno-deficiency to Epstein-Barr virus infection that has been mapped to chromosome Xq25. Molecular analysis of XLP patients from ten different families identified a small interstitial constitutional deletion in 1 patient (XLP-D). This deletion, initially defined by a single marker, DF83, known to map to interval Xq24-q26.1, is nested within a previously reported and much larger deletion in another XLP patient (XLP-739). A cosmid minilibrary was constructed from a single mega-YAC and used to establish a contig encompassing the whole XLP-D deletion and a portion of the XLP-739 deletion. Based on this contig, the size of the XLP-D deletion can be estimated at 130 kb. The identification of this minimal deletion, within which at least a portion of the XLP gene is likely to reside, should greatly facilitate efforts in isolating the gene.

Trapping of mammalian promoters by Cre-lox site-specific recombination.

One of the challenges in human genome research is to identify the promoter sequences which play a key role in the regulation of gene expression. We report here a new promoter trapping system for use with mammalian cells comprised of the following three steps: 1) Cloning of DNA fragments into a promotertrapping vector, 2) integration of the trapping vector into a designated target in the mammalian genome using the Cre site-specific recombinase, and 3) screening of integrants for trapped promoter sequences by activation of the luciferase gene. To assess the efficiency of this system, lox trapping vectors containing sense tk promoter, antisense tk promoter, or a non-promoter sequence of the neo gene were employed. The resulting levels of luciferase activity of the site-specific integrants were measured directly. Luciferase activity of the integrants can be assayed under conventional culture conditions by simply replacing the culture medium with potassium phosphate buffer containing luciferin. Only those G418r colonies carrying the tk promoter in the normal orientation exhibited a 21-to 35-fold increase in luciferase activity over that of the other integrants. These results indicate that this system is an effective means of trapping promoter sequences from random mammalian genomic DNA fragments.

The primary structure and genomic organization of five novel transcripts located close to the Huntington's disease gene on human chromosome 4p16.3.

Five distinct novel transcripts (RES4-22, -23, -24, -25 and -26) that mapped to the 1-Mb interval between D4S180 and D4S183 on human chromosome 4p16.3 close to the Huntington's disease (HD) gene were isolated, and the structure and exon/intron organization of each gene were thoroughly analyzed. The transcripts of the RES4-22, -23 and -24 genes each have several isoforms by alternative splicing and these have also been defined. Two transcripts, RES4-24 and RES4-25, reside in the same genomic region with opposite polarities and they also clearly overlap. Among these transcripts, RES4-26 was found to encode a novel zinc finger protein. The transcript map based upon our current level of analysis combined with data from previous studies reveals the gene-rich nature and the intricate organization of the genes in the HD locus.

Transcript map of the human chromosome 4p16.3 consisting of 627 cDNA clones derived from 1 Mb of the Huntington's disease locus.

Six hundred and twenty-seven cDNA clones from human brain cDNA libraries were characterized and integrated into a transcript map of the 1-Mb region on human chromosome 4p16.3 containing the Huntington's disease (HD) gene. Six hundred and seventy-two cDNA clones were obtained by a direct screening of the cDNA libraries, probing with pools of single copy microclones generated from the HD region specific yeast artificial chromosome (YAC)-DNA. So far, 93% of the obtained clones (627 cDNA clones) have been mapped onto the 1-Mb HD gene region by hybridization with HD region-specific cosmid, P1 and YAC clones. DNA sequence and expression analyses revealed that several cDNA clones might encode novel genes, some of which are situated within or close to the IT15, IT11, and alpha-adducin (ADD1) gene region, suggesting the presence of the overlapping genes in this region. This collection of cDNA clones will greatly facilitate the construction of the complete map of the transcripts in the HD region.

The brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy: an MRI study.

We studied the frequency and characteristics of brainstem and thalamic lesions in dentatorubral-pallidoluysian atrophy using MRI. Of 15 subjects diagnosed by DNA analysis, 13 had lesions in the pontine base, nine in the midbrain, and five in the thalamus. Lesions were correlated positively with the patient's age, but not with neurologic features or numbers of CAG repeats. Patients with Machado-Joseph disease or spinocerebellar ataxia 1 did not show these characteristic lesions.

RNA polymerase in vegetative cells of Bacillus subtilis. I. Purification and properties of RNA polymerase L1 and L2.

Two forms of RNA polymerase [EC 2.7.7.6], RPase L1 and RPase L2, isolated from a highly synchronized vegetative culture of Bacillus subtilis Marburg strain are described. RPase L1 is the major component (identical and the vegetative RNA polymerase already reported) and RPase L2 is a minor, new component corresponding to 3--5% of the total activity. The enzymes differed in their requirements for divalent ions, though the differences depended on the template DNA employed. PRase L1 is able to transcribe phage M2 DNA in the presence of Mg2+ ions and both B. subtilis DNA and phage M2 DNA in the presence of Mn2+ ions. On the other hand, RPase L2 activity can be detected only in the presence of 3 mM Mn2+ ions with all the templates. It is of interest that the transcription of phage M2 DNA by both enzymes stringently requires KC1. It may be due to this ion dependence that RPase L2 has not been detected previously. RPase L2 consists of 1beta', 1beta gamma, 1sigma, and 2alpha subunits. The molecular weight of the beta gamma subunit (about 110,000) is close to the value reported for the beta subunit of RNA polymerase prepared from sporulating cells. However, RPase L2 as a whole molecule is different from the RNA polymerase of sporulating cells or spores in the following two respects: RPase L2 contains sigma subunit as a component essential for selective transcription, and it is resistant to 1 mug of rifampicin per ml. Elimination of the sigma subunit from RPase L2 greatly stimulates RNA synthesis by the enzyme. Conversely, the addition of sigma subunit to the core-enzyme is inhibitory.

RNA polymerase in vegetative cells of Bacillus subtilis. II. New polypeptide factors, FI and FII, stimulating in vitro RNA synthesis directed by phage M2 DNA.

Two polypeptides named FI and FII were isolated from vegetative cells of Bacillus subtilis Marburg. The molecular weights of FI and FII were 15,000 and 30,000 daltons, respectively. They were able to stimulate the transcription of phage M2 DNA in the presence of Mg2+ ions by RPase L1 and RPase L2 [RNA polymerase; EC 2.7.7.6]. Although both core- and holo-RPase L2 hardly exhibited transcription activity under these conditions, the factors could stimulate both activities up to the level of RPase L1 activity. The stimulation was much less marked when B. subtilis DNA was used as a template. These stimulatory functions were found to lie not in the chain elongation but in the initiation step of transcription, following the preinitiation step. To obtain stimulation by the factors, preincubation with RNA polymerase was necessary. FI stimulated RPase L1 or RPase L2 only when preincubated in the stimultaneous presence of FII, forming a complex, RPase L1(or L2)-FI-FII. On the other hand, FII alone could stimulate transcription, forming a complex. RPase L1 (or L2)-FII. In these complexes, the ratio of FI, FII, and RPase L1(or L2) was 1 : 1: 1. Although the core-RPase L2 activity was inhibited by sigma subunits, it was not inhibited by was rather stimulated when the enzyme was present as a complex with FI and FII. Thus the complex, consisting of RPase L2 and the factors, resembled RPase L1 with respect to molecular weight, template specificity, the effect of sigma subunit, and sensitivity to rifampicin.

[Laser chromosome microdissection and cloning of the genetic disease loci].

We have developed an argon ion laser chromosome microdissection technique in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) to directly amplify microdissected chromosomes. The 22-mer primer used in PCR, although unique in sequence, randomly primed and amplified any target DNA. These methods were applied to both the terminal region of the human chromosome 4p (4p 16) and Xq (Xq26-q28), and two chromosome region-specific DNA libraries were constructed. The resulting libraries contained approximately 1000 nonoverlapping DNA sequences with an average size of 230-350 bp, at an average spacing of 10-65 Kbp along the chromosomes of origin. Our new method is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.

The position end-set tree: a small automaton for word recognition in biological sequences.

We consider the basic function which locates a specific string of symbols within a longer sequence. When one is expecting to do many substring searches it is worthwhile to build an auxiliary index to the sequence to aid in the search. We propose a method to generate a compact index that can be viewed as a small (partial) deterministic finite automaton recognizing the subword structure of a sequence. We present an algorithm for its construction on-line in linear time. Such a data structure permits the efficient localization of subwords in a sequence and can be used in the development of interactive sequence analysis software.

Pattern recognition in DNA sequences and its application to consensus foot-printing.

We consider the problem of comparing several nucleic acid sequences to identify words occurring imperfectly (patterns with no gap) with unusual frequency. Methods for computing, representing, and inspecting interactively the structure of such repeating motifs in nucleic acids and more generally any text are described. Multiple sequences are treated as one large concatenate. In a preprocessing step, a lexical index is created to provide rapid string matching for the enumeration of the words matching a pattern. For given word features (word length, minimal frequency), a sequence profile is displayed. The profile can be inspected interactively with on-line algorithms. Applications to the identification of regulatory elements in DNA regions involved in the control of gene expression are presented. Our program ('DNA-Lexemics') runs on the Macintosh.

A small automaton for word recognition in DNA sequences and its application to consensus analysis of regulatory elements in DNA regions controlling gene expression.

A method for pattern analysis of DNA sequence data is considered. A space economical automaton for word recognition was presented elsewhere together with an algorithm for its compilation in linear time. An algorithm for the localization of words including imperfect matches (motif search) was developed. A program was implemented on the Macintosh and used extensively for the representation of the word composition of DNA data. We explore different sets of regulatory sequences to illustrate the performance of this method. In mammalian DNA, this analysis reveals "consensus motifs" corresponding to functional (or putative) cis-acting elements mediating the regulation of gene expression.

A fast word search algorithm for the representation of sequence similarity in genomic DNA.

Representation of sequence similarity by dot matrix plots is a method widely used for comparing biological sequences. The user is presented with an overall view of similarity between two sequences. Computation of this plot has been reconsidered here. An improvement is proposed through the preprocessing of the data into an automation recognizing the word structure of a sequence. The main advantage of this approach is to systematically eliminate the repetitions during word comparison. Simple heuristics are also considered to greatly speed up pattern matching. As a result, large sequences are handled very efficiently. This is illustrated by a comparison of large genomic DNA. The algorithm has been implemented in an interactive application on a microcomputer.

The structures of integration sites in transgenic rice.

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT-rich region and/or at the repetitive sequence region, including a SAR-like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.

Replication of adenovirus DNA-protein complex with purified proteins.

A protein fraction isolated from the cytosol of adenovirus-infected HeLa cells, which contained DNA polymerase alpha, catalyzed adenoviral DNA replication in the presence of adenovirus DNA binding protein, eukaryotic DNA polymerase beta, ATP, all four dNTPs, and MgCl2. DNA replication started at either end of exogenously added adenoviral DNA and was totally dependent on the presence of terminal 55,000-dalton proteins on the DNA template. The replicaton of adenovirus DNA in the system was sensitive to aphidicolin and retained nearly all the properties of DNA replication that occur in vivo or in vitro with crude extracts. The 5'-ends of the newly synthesized adenovirus DNA strands were covalently linked to an 80,000-dalton protein linked to dCMP. DNA synthesized with purified proteins was only 25-50+ the length of parental viral strands. Addition of cytosol extracts from uninfected HeLa cells to reaction mixtures containing purified proteins gave full-length adenoviral DNA strands.

Role of polymeric forms of the bacteriophage phi X174 coded gene A protein in phi XRFI DNA cleavage.

Gene A of the phi X174 genome codes for two proteins, A and A* (Linney, E.A., and Hayashi, M.N. (1973) Nature New Biol. 245, 6-8) of molecular weights 60,000 and 35,000, respectively. The phi X A* protein is formed from a natural internal initiator site within the A gene cistron while the phi X A protein is the product of the entire A gene. These two proteins have been purified to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Previous studies have shown that the phi X A protein is an endonuclease which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted phi XRFI DNA. In addition to this activity, the phi X A protein also causes relaxation of supertwisted phi XRFI DNA and formation of a phi XRFH DNA . phi X A protein complex which has a discontinuity in the A cistron of the viral strand. This isolatable complex supports DNA synthesis when supplemented with extracts of uninfected Escherichia coli which lack phi X A protein and phi XRFI DNA. The phi XRFII DNA . phi X A protein complex can be attacked by exonuclease III but is not susceptible to attack by E. coli DNA polymerase I, indicating that the 5'-end of the complex is blocked. Attempts to seal the RFII structure generated from the phi XRFII DNA . phi X A protein complex with T4 DNA ligase in the presence or absence of DNA polymerase were unsuccessful. The phi X A protein does not act catalytically in the cleavage of phi XRFI DNA. Under conditions leading to the quantitative cleavage of phi XRFI DNA, the molar ratio of phi XRFI DNA to added phi X A protein was approximately 1:10. At this molar ratio, cross-linking experiments with dimethyl suberimidate yielded 10 distinct protein bands which were multiples of the monomeric phi X A protein. In the absence of DNA or in the presence of inactive DNA (phi XRFII DNA) no distinct protein bands above a trimer were detected. We found it possible in vitro to form a phi XRFII DNA . phi X A protein complex with wild-type phi XRFI DNA (phi X A gene+) and with phi XRFI DNA isolated from E. coli (su+) infected with phage phi X H90 (an am mutant in the phi X A gene). Thus, in vitro, in contrast to in vivo studies, phi X A protein is not a cis acting protein. The purified phi X A* protein does not substitute for the phi X A protein in in vitro replication of phi XRFI DNA nor does it interfere with the action of the phi X A protein which binds only to supertwisted phi XRFI DNA. In contrast, the phi X A* protein binds to all duplex DNA preparations tested. This property prevents nucleases of E. coli from hydrolyzing duplex DNAs to small molecular weight products.

Adenoviral protein-primed initiation of DNA chains in vitro.

The initiation of DNA chains by the 80-kilodalton form of the adenovirus terminal protein has been studied. This protein, which can be covalently linked to dCMP, is isolated complexed to a 140-kilodalton protein possessing DNA polymerase activity. In the presence of adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex requires the addition of ATP and nuclear extract from uninfected cells in addition to Mg2+ and dCTP. When single-stranded DNA is used in place of the adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex occurs in the absence of ATP and nuclear extract. In the presence of the four dNTPs, the complex yields DNA chains of various sizes between 100 and 300 nucleotides. The products formed with bacteriophage phi X174 single-stranded circular DNA as the template are site specific, predominantly derived from the sequences between nucleotides 2363 and 2977 and between nucleotides 3760 and 4206. These small dNA chains are blocked at their 5' ends with the 80-kilodalton protein but possess free 3'-OH ends that are susceptible to degradation by exonuclease III and can be elongated to replicative form II products with DNA polymerase I of Escherichia coli or eukaryotic DNA polymerase beta preparations. A protein priming model explaining the different requirements for initiation with adenovirus DNA-protein and with phi X174 DNA is presented.