ABI 373A Research Articles

Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2.

Identification of a Novel Protein with a Role in Lipoarabinomannan Biosynthesis in Mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.

Rapid communication: nucleotide sequence of the ovine lipoprotein lipase cDNA.

Name of the Sequence. Ovine lipoprotein lipase cDNA. Genus and Species. Ovis aries. Origin of Clones. Because almost the whole 3′-untranslated region (3′UTR) was found to be lacking from the ovine lipoprotein lipase (LPL) cDNA sequence (1,656 bp including the 5′UTR, the coding sequence, and the first 44 bp of the 3′UTR) published by Edwards et al. (1993), we have undertaken to characterize this region, with the aim to provide a full-length LPL cDNA. First, a 779-bp LPL 3′UTR cDNA was obtained from ovine adipose tissue by rapid amplification of cDNA end (3′RACE) using the following primers: forward 5′-GTATAGTGGCCAAATAGCACA-3′, reverse 5′-TCAAGCTTCTGCAGGATCCTTTTTTTTT TTTTTTTT-3′. The reverse primer was also used to prime reverse transcription. The RACE products were cloned into SmaI-digested and dephosphorylated pGEM-4Z. The LPL recombinant plasmids were screened by PCR performed directly on colonies. Second, the sequence of the fragment lacking between the previous 779-bp fragment and the fragment sequenced by Edwards et al. (1993) was amplified by PCR with the forward 5′-CAAGTCTCTGAATAGAAAGTCT-3′ and reverse 5′-GATTTCCAGTAATAGCCTCTG-3′ primers. Sequencing reactions were carried out on both strands, using an ABI 373A automated DNA sequencer and the accompanying software (PE Applied Biosystems, Courtaboeuf, France).

New microsatellite markers for assessment of paternity in the squid Loligo forbesi (Mollusca: Cephalopoda).

Correspondence: P. Escobar-Paramo; Fax: +1-516 6327626; E-mail: pacopa@life.bio.sunysb.edu Molecular ecological studies are not common in primatological research. Part of the problem with monkeys from Central and South America (New World) is the difficulty of obtaining DNA from the animals, given their arboreal life. This prob- lem can now be overcome with the new methods developed to extract DNA from fecal material, food wedges and urine. The second problem is the lack of microsatellite primers that can be used to amplify the small amounts of DNA found in the fecal samples. Here, I report the characterization of five microsatellite primers isolated from Cebus apella, and tv\ro primers isolated from Lagothrix lagotricha. The use of these primers in other neotropical primate species is also reported. Five micrograms of genomic DNA from Cebus apella and Lagothrix lagotricha were digested with Sau3AI and frag- ments in the range of 400-1000 bp were selected for cloning into pBluescript-SK cut with Bamiil. Eight-hundred colonies of each species were screened with (GT)jg and (CT)jg DIG-labeUed probes and positive clones were detected using a DIG detection kit (Boehringer Mannheim). Positive clones were sequenced on an Applied Biosystem ABI 373A automated sequencer, and analysed using Perkin Elmer DNA Sequencing Software (version 2.1.1). Out of the five clones sequenced from the Lagothrix library, four contained microsatellites, and out of the 13 clones sequenced from the Cebus hbrary, nine con- tained microsatelhtes. Primers were designed using GeneRun software. Seven of the 14 pairs of primers designed are reported in Table 1. PCR conditions were optimized on Cebus DNA extracted from blood using standard phenol-chloroform extrac- tions. DNA amplifications were performed in a total volume of 25 |xL (10 mM Tris-HCl (pH 8.9), 50 mM KCl, 20

Duplications and de novo deletions of theSMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence‐based carrier testing for spinal muscular atrophy

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances. Am. J. Med. Genet. 85:463–469, 1999. © 1999 Wiley-Liss, Inc.

Forensic validation of the short tandem repeat HUMACTBP2 using capillary electrophoresis.

Experiments were performed to evaluate the forensic identification of the short tandem repeat (STR) HUMACTBP2 (human beta-actin-related pseudogene) using automated fluorescence-based capillary electrophoresis. The HUMACTBP2 is a complex tetranucleotide STR locus with more than 32 alleles in the range of 202-323 bp. The reproducibility of genetic typing using a fluorescent labeled allelic ladder was determined by comparison of the calculated fragment size after consecutive (within-day) and nonconsecutive (day to day) injection. The maximum variation in size (window) observed for any allele was 0.23 bp for the within-day and 0.8 bp for the day-to-day precision. Furthermore, it is possible to achieve a 1 bp resolution, the precision of the reproducibility assays being about 99.95%. Sixty blood samples and twenty stains were typed with both automated fluorescent sequencer ABI 373A and ABI 310. Identical genotypes were obtained with both techniques and the ABI 310 seemed to be more sensitive than the ABI 373A. A population sample of 197 unrelated individuals from southwest Switzerland was analyzed and the genotype frequencies observed were similar to those reported by others. Thirty-one alleles and 126 genotypes were found. The observed heterozygosity was 0.934. Mixtures from two different blood samples varying in their ratio were typed and the minor fraction was detectable to about 1:10. The practical usefulness of the HUMACTBP2 is illustrated by analyzing casework samples. This validation study proves the usefulness of the HUMACTBP2 locus in forensics and the detection efficiency using fluorescent capillary electrophoresis.

A simple method for automated allele binning in microsatellite markers.

High-throughput fluorescent genotyping requires a considerable amount of automation for accurate and efficient processing of genetic markers. Automated DNA sequencers and corresponding software products are commercially available that contribute substantially to increased throughput rates for large-scale genotyping projects. However, some conceptually simple tasks still require time-consuming manual intervention that imposes bottlenecks on throughput capacity. One of these tasks is the conversion of imprecise DNA fragment sizes determined by commercial software programs to the underlying discrete alleles that the sizes represent. Here we describe a simple method for assigning allele sizes into their appropriate allele "bins" using least-squares minimization procedures. The method requires no special treatment of family data on plates, internal/external size standards, or electropherogram data manipulation. Tests of the method using the ABI 373A automated DNA sequencer and accompanying Genescan/Genotyper software resulted in accurate automatic classification of all alleles in >80% of 208 markers analyzed, with the remaining 20% being appropriately identified as requiring additional attention to laboratory conditions. Specific characteristics of different markers, including differences in PCR product size and inexact repeat lengths (e.g., 1. 9 bp for a dinucleotide repeat), are accommodated by the method and their properties discussed.

Analysis of triplet repeats of the FRAXA locus using a novel sequencing procedure.

Recent reports have shown that in normal alleles, the CGG repeat at the FRAXA locus is interrupted by one or two AGG and that abnormal alleles seem to be generated by expansion of pure CGG repeats at the 3′ end [1].It is therefore important to establish rapid, simple and low-cost sequencing procedures for determining not only the number of CGG repeats but also the presence of AGG triplets.A one-lane sequencing procedure with PCR-amplified DNA, labelled at the 3′ or 5′ end with a single fluorochrome, has been recently developed in our laboratory [2, 3]. This methodology is particularly suitable for detecting mutations in family studies [4] and has been applied to the analysis of the sequence of triplet repeats at the FMR1 gene after PCR amplification, using one of the two primers fluorescently labelled at the 5′ end.The sequence of the primers utilised in the PCR reaction is reported by Erster et al. [5]. The direct primer was fluorescently labelled at the 5′ end with 5-(6)-carboxyfluorescein (Fluoreprime, Pharmacia) and synthesised using an ABI 391 PCR-Mate-EP DNA synthesiser. The PCR amplification was performed on 0,5 ug DNA in a 50-μ1 PCR buffer containing 10% DMSO, 10% glycerol, 25 pmol of the two primers, 0,3 mM dNTPs and 1 U Taq DNA polymerase.Each sample was submitted to amplification on a Perkin Elmer 9600 PCR reactor, with the following cycling profile: 5 min at 95 °C, 30 cycles of 30 s at 97 °C, 1 min at 55 °C and 1 min at 72 °C, followed by a 10-min terminal extension at 72 °C. After precipitation with 5 M ammonium acetate, the pellet was dissolved in 80% (vol/vol) formamide and aliquots were heated for 2 min at 90 °C or, alternatively, for sequence analysis, for 10 min at 110 °C. The samples were loaded on 6 or 8% PAGE sequencing gels and analysed using an ABI 373A automatic sequencer as previously described [2].

Correction of Some Genotyping Errors in Automated Fluorescent Microsatellite Analysis by Enzymatic Removal of One Base Overhangs

The conjunction of high resolution genetic maps based on (CA)n microsatellite markers (1) and fluorescent genotyping (2) has led to research programs which require the determination of hundreds of thousands of genotypes. However, the migration profile of a (CA)n microsatellite marker after PCR (polymerase chain reaction) is often complicated because of slippage of Taq polymerase during PCR, and because of the ability of this polymerase to add an extra base at the end of the amplified fragments in a template dependent manner (3). In the traces generated by a fluorescent sequencer, these phenomena generate spurious bands as shown in Figure 1. These extra bands are usually referred to as ‘shadow bands’ (4). This complicated pattern is relatively easily recognized by an experienced eye, but may mislead the software which automatically determines the sample alleles (we use ABI 373A sequencers with 24 cm gels for the electrophoresis, and the ABI software Genescan and Genotyper for peak sizing and allele calling respectively). Because of this, the operator must check visually all the traces and the allele calls made by the software. This is time-consuming, and prone to human error after a few hundred checks. We attempted therefore to eliminate most of the allele calling errors by eliminating the extra bases added by the Taq polymerase. We tried to do this by using Pfu polymerase either during or after PCR, or by using T4 DNA polymerase after PCR. Pfu polymerase is thermostable and possesses a proofreading activity (3). In some cases, however, the extra base added by the Taq polymerase was not completely removed by this enzyme (Fig. 2A and B). This phenomenon is probably due to competition between the extendase activity of the Taq polymerase and the exonuclease activity of the Pfu polymerase as both enzymes are active at 72 C. On the contrary, and as suggested by others (5), a T4 polymerase treatment, similar to that conventionally used to make blunt double-stranded linear DNA fragments, completely removed the bases added by the Taq polymerase (without exception so far; Fig. 2C). The simplification of the electrophoresis pattern is sometimes spectacular (Fig. 3). The use of Pfu polymerase alone during PCR avoids the problem of the extra base addition (3), and was used successfully in fluorescent genotyping (6). However, PCR efficiency is lower than when using Taq polymerase, making it necessary to precipitate the samples during multiplex analysis (data not shown). Figure 1. Typical electrophoresis pattern of a (CA)n microsatellite marker with shadow bands. The two alleles of this heterozygous sample are eight bases apart, and are shown with their estimated size. The rightmost peak of each group corresponds to an extra base addition (peaks labelled +1). On the left, we see peaks corresponding to slippage of the Taq polymerase (labelled –2 and 4), and the ‘+l peaks’ of these ‘slippage peaks’. For this marker, the normal peaks and the +l peaks have a similar height. That means that, depending on the samples, the Genotyper software will choose one peak or the other for the allele, leading to errors in the genetic data.

Fluorescent multiplex microsatellites used to define haplotypes associated with 75 CFTR mutations from the UK on 437 CF chromosomes.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene contains three highly informative microsatellites: IVS8CA, IVS17bTA, and IVS17bCA. Their analysis improves prenatal/ carrier diagnosis and generates haplotypes from CF chromosomes that are strongly associated with specific mutations. Microsatellite haplotypes were defined for 75 CFTR mutations carried on 437 CF chromosomes (220 for delta F508, 217 for other mutations) from Northern Ireland and three English regions: the North-West, East Anglia, and the South. Fluorescently labelled microsatellites were amplified in a triplex PCR reaction and typed using an ABI 373A fluorescent fragment analyser. These mutations cover all the common and most of the rare CF defects found in the UK, and their corresponding haplotypes and geographic region are tabulated here. Ancient mutations, delta F508, G542X, N1303K, were associated with several related haplotypes due to slippage during replication, whereas other common mutations were associated with the one respective haplotype (e.g., G551D and R560T with 16-7-17, R117H with 16-30-13, 621 + 1G > T with 21-31-13, 3659delC with 16-35-13). This simple, fast, and automated method for fluorescent typing of these haplotypes will help to direct mutation screening for uncharacterised CF chromosomes.

Locus ACTBP2 (SE33). Sequencing data reveal considerable polymorphism.

A total of 50 different ACTBP2 (human beta-actin related pseudogene) alleles and the cell line K562 were sequenced and analysed. Sequence data revealed not only length polymorphism but also a highly variable sequence polymorphism. 2 different sequence structures were characterised. Type I (223-259bp) contained the known regular 4bp repeat AAAG. Type II (265-309bp) revealed a further hexanucleotide unit AAAAAG in addition to the common AAAG which only occurs once in the repeat region. The position of this insertion showed considerable variation. To obtain a regular spaced allelic ladder 20 of the sequenced alleles were selected. Denaturing gels and high resolution/non-denaturing gels were compared and striking differences could be seen between the 2 gel systems. Separation of the alleles on a 6% denaturing gel and analysis on the ABI 373A Sequencer revealed fragment sizes which corresponded to the sequencing data but were in general 6-10bp longer. In contrast, in non-denaturing gels some alleles showed different electrophoretic mobilities compared to the sequenced allelic ladder which could indicate different fragment length and/or different sequence structure.

Quantification of mRNA in Human Tissue Using Fluorescent Nested Reverse-Transcriptase Polymerase Chain Reaction

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/μg total RNA. In medulla these values were 0.46 and 1.16 amol/μg, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Strategies for rapid and accurate DNA sequencing

In an effort to increase the amount of accurate final sequence data, we now report our latest template DNA isolation procedure, fluorescent and radiolabeled DNA sequencing reactions, physical modifications to the ABI 373A fluorescent-based DNA sequencer, and the development of a new fluorescent data analysis program that results in manually interpretable sequence data in excess of 750 nucleotides from the priming site in a single reaction. In addition, a sequencing strategy that combines these isolation and sequencing methods with initial shotgun cloning followed by a short oligonucleotide primer walking approach for both contig joining and proofreading is described. When combined, these methods and strategies significantly increase the rate and ease of DNA sequence data acquisition, as well as improve the extent and accuracy of the sequence data.

A trace display and editing program for data from fluorescence based sequencing machines

'Ted' (Trace editor) is a graphical editor for sequence and trace data from automated fluorescence sequencing machines. It provides facilities for viewing sequence and trace data (in top or bottom strand orientation), for editing the base sequence, for automated or manual trimming of the head (vector) and tail (uncertain data) from the sequence, for vertical and horizontal trace scaling, for keeping a history of sequence editing, and for output of the edited sequence. Ted has been used extensively in the C.elegans genome sequencing project, both as a stand-alone program and integrated into the Staden sequence assembly package, and has greatly aided in the efficiency and accuracy of sequence editing. It runs in the X windows environment on Sun workstations and is available from the authors. Ted currently supports sequence and trace data from the ABI 373A and Pharmacia A.L.F. sequencers.