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PubMed2005

Rim 2/Hipa CACTA transposon display: a new genetic marker technique in Oryza species.

BACKGROUND: Transposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa-TD) protocol for the study of genetic markers in map construction and the study of genetic diversity in rice. RESULTS: Rim2/Hipa-TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa-TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa-TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa-TD. While Rim2/Hipa-TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC, EE, BBCC and CCDD, profiles demonstrated that most of the amplified fragments were larger than 400 bp, and that our methods were insufficient to clearly distinguish between these fragments. However, the overall amplification profiles between species in the Oryza genus were fully distinct. Phenetic relationships among the AA diploid Oryza species, as evidenced by the Rim2/Hipa-TD markers, were matched with their geographical distributions. CONCLUSION: The abundance of the Rim2/Hipa TIR sequences is very informative since the Rim2/Hipa-TD produced high polymorphic profiles with ample reproducibility within a species as well as between species in the Oryza genus. Therefore, Rim2/Hipa-TD markers can be useful in the development of high-density of genetic map around the centromeric regions. Rim2/Hipa-TD may also prove useful in evaluations of genetic variation and species relationships in the Oryza species.

Base Sequence↗

PubMed2001

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria.

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Amino Acid Sequence↗

PubMed1988

Structure and expression of rodent genes encoding the testis-specific cytochrome c. Differences in gene structure and evolution between somatic and testicular variants.

Mammalian testis contains two forms of cytochrome c, one identical to the form found in somatic tissues and a second that is expressed in a stage-specific manner during spermatogenic differentiation. We have isolated both rat and mouse cDNA clones and the rat gene encoding the testis-specific cytochrome c and determined their DNA sequences. The testicular variant displays a number of notable differences with its somatic counterpart. 1) In contrast to the multipseudogene family derived from mammalian somatic cytochrome c genes, the testis gene is single-copy in genomic DNA with no detectable pseudogenes. 2) The rat testis gene is approximately 7 kilobases (kb) long with three introns totaling nearly 6.5 kb whereas the two introns dividing the 2.1-kb somatic gene occupy only 0.9 kb. Introns differ in position as well as size. 3) The testicular variant has a longer 5'-untranslated leader (230 versus 70 base pairs for the somatic gene) with an upstream open reading frame of 129 base pairs beginning with an AUG in a favorable translational context. 4) A single polyadenylation site in the testicular mRNA (approximately 900 nucleotides) contrasts with the three functionally equivalent sites observed in rat somatic messages. 5) Finally, rat and mouse testis cytochromes c differ at 4 amino acid residues as opposed to the complete sequence identity found in the somatic proteins suggesting a shorter unit evolutionary period for these molecules. These observations are consistent with a duplication of an ancestral cytochrome c gene leading to the emergence of novel structural features and regulatory properties likely associated with the striking tissue specificity of the testicular cytochrome c.

Amino Acid Sequence↗

PubMed1999

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.

Chromosomes, Human↗

PubMed2004

Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.

BACKGROUND/AIMS: Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner. METHODS: The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif. RESULTS: The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance. CONCLUSIONS: The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy.

Amino Acid Motifs↗

PubMed2003

Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains.

Mutations that disrupt a PY motif in epithelial Na+ channel (ENaC) subunits increase surface expression of Na+ channels in the collecting duct, resulting in greater Na+ reabsorption. Recently, Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4-2 (hNedd4-2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5'-exons were identified by 5'-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3'-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4-2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4-2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+ transport regulation. In vitro binding studies demonstrated that individual PY motifs of alpha-, beta-, and gamma-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4-2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4-2 in the regulation of ENaC and define protein domains important for Nedd4-2 function.

3' Untranslated Regions↗

PubMed1996

[Evolution modes of eukaryote retrotransposons].

A number of general problems of molecular macroevolution of retroposons were examined, including the question of the ratio of the contributions of genomic replication and retrotransposition to mutational variability of retrotransposons, as well as that of the influence of stress-induced transpositional variability on the rate of evolution and the phylogenetic trees of retrotransposons. It is thought that the substitution fixation rate in genes of retrotransposons is determined by the transposition rate and the probability of mutation upon replication of copies of retransposons in the genome and upon retrotransposition, as well as by selection stabilizing the function of proteins of the retroreplicative mechanism. By means of molecular-evolutionary parameters, estimated for Drosophila retrotransposons and animal retroviruses, it was shown that spontaneous retrotransposition makes the dominant contribution to the frequency of mutation, and the expected fixation rate of nucleotide substations is on the order of 2 x 10(-8) per position per year. This version of evolution of retrotransposons agrees with the results of phylogenetic analysis of trees of macroevolution. Another version, associated with stress-induced transpositions, gives a very high rate of evolution, on the order 3 x 10(-6) fixations of nucleotide substitutions per position per year. This version seems improbable, as it leads to a significant hidden genetic load.

Animals↗

PubMed1992

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.

Animals↗

PubMed2004

Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements.

BACKGROUND: The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. RESULTS: We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. CONCLUSIONS: Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers.

Alu Elements↗

PubMed2004

Tools for loading MEDLINE into a local relational database.

BACKGROUND: Researchers who use MEDLINE for text mining, information extraction, or natural language processing may benefit from having a copy of MEDLINE that they can manage locally. The National Library of Medicine (NLM) distributes MEDLINE in eXtensible Markup Language (XML)-formatted text files, but it is difficult to query MEDLINE in that format. We have developed software tools to parse the MEDLINE data files and load their contents into a relational database. Although the task is conceptually straightforward, the size and scope of MEDLINE make the task nontrivial. Given the increasing importance of text analysis in biology and medicine, we believe a local installation of MEDLINE will provide helpful computing infrastructure for researchers. RESULTS: We developed three software packages that parse and load MEDLINE, and ran each package to install separate instances of the MEDLINE database. For each installation, we collected data on loading time and disk-space utilization to provide examples of the process in different settings. Settings differed in terms of commercial database-management system (IBM DB2 or Oracle 9i), processor (Intel or Sun), programming language of installation software (Java or Perl), and methods employed in different versions of the software. The loading times for the three installations were 76 hours, 196 hours, and 132 hours, and disk-space utilization was 46.3 GB, 37.7 GB, and 31.6 GB, respectively. Loading times varied due to a variety of differences among the systems. Loading time also depended on whether data were written to intermediate files or not, and on whether input files were processed in sequence or in parallel. Disk-space utilization depended on the number of MEDLINE files processed, amount of indexing, and whether abstracts were stored as character large objects or truncated. CONCLUSIONS: Relational database (RDBMS) technology supports indexing and querying of very large datasets, and can accommodate a locally stored version of MEDLINE. RDBMS systems support a wide range of queries and facilitate certain tasks that are not directly supported by the application programming interface to PubMed. Because there is variation in hardware, software, and network infrastructures across sites, we cannot predict the exact time required for a user to load MEDLINE, but our results suggest that performance of the software is reasonable. Our database schemas and conversion software are publicly available at http://biotext.berkeley.edu.

Database Management Systems↗

PubMed1998

[Cytogenetic of nucleolar organizer regions (NOR) of human chromosomes: identification of four morphofunctional variants of NOR, their inter-individual and inter-chromosomal distribution].

Cytogenetic characters of the nucleolus organizer regions (NORs) located on the short arms of five acrocentric chromosomes were studied in chromosome preparations obtained from cultured blood cells of 17 donors. In situ hybridization to a 3H-labeled probe was used to estimate the relative copy number of ribosomal genes (RGs) in all NORs of chromosomes identified by G-banding in each sample. The relative amount of potentially active RGs (0 to 4 arbitrary units) in each NOR was estimated from the size of AgNOR selectively stained with silver nitrate. Linear regression analysis revealed clusters of silent RGs (CSRGs) in 24 out of 170 NORs (14%). Based on the presence or absence of active and inactive RG clusters, NORs of human chromosomes were classified into four morphological functional variants (MFVs): (1) Ag-/CSRG-, (2) Ag-/CSRG+, (3) Ag+/CSRG-, and (4) Ag+/CSRG+. These variants were observed in 7.65%, 2.35%, 11.8%, and 78.2% of 170 analyzed NORs, respectively. NORs with CSRGs (MFV 2 and 3) were absent in 5 out of 17 donors. One, two, and three NORs with CSRGs were observed in four donors each. Analysis of the chromosome distribution of NOR MFVs showed that their frequencies remained almost the same in group-D and group-G acrocentric chromosomes. Although the tested samples were small (34 chromosomes for each pair), two observations were made with regard to individual chromosomes. First, almost half MFV-1 NORs (6 out of 13) were detected on chromosome 15. Second, the frequency of CSRGs was higher in chromosome 21 (29%) than in the other chromosomes (10%).

Adult↗

PubMed2000

[A study of the genetic diversity of common wild rice (O. rufipogon Griff.) and cultivated rice (O. sativa L.) by RFLP analysis].

The genetic diversity of cultivated rice and common wild rice from 10 countries in Asia were evaluated by RFLP analysis using 44 probes of single copy. We used the parameters such as proportion of polymorphic loci (P), average number of alleles per locus (A), average number genotype per locus (Ng), degree of heterozygate per locus (Ho) and average gene diversity (Hs) showing genetic diversity. It is indicated that the common wild rice from China has the largest genetic diversity in the 10 countries, and the secondary is the common wild rice from India. The results also showed that the genetic diversity in Chinese common wild rice is larger than the genetic diversity in common wild rice of South and Southeast Asia. The average gene diversity (Hs) of South Asian wild rice is higher than Southeast Asian wild rice, while the parameters value of proportion of polymorphic loci (P), average number of alleles per locus (A) and average number genotype per locus (Ng), Southeast Asian wild rice is higher than South Asian wild rice. It is found that the genetic diversity in cultivated rice is obviously lower than in common wild rice. In the detected 44 loci, the number of polymorphic loci on O. sativa is as the same as 3/4 in O. rufipogon, the number of alleles in O. sativa is as the same as 60% in O. rufipogon, the number of genotype in O. sativa is as same as 1/2 in O. rufipogon. In O. sativa, the genetic diversity of indica is larger than that of japonica. O. rufipogon from China have highest value in degree of hetrozygate per locus (Ho), and Ho of wild rice is as two times as cultivated rice. It is showed that in the proceeding of wild rice evolved into cultivated rice, Ho and number of alleles was reduced, and the genetic diversity was decreased.

Genetic Variation↗

PubMed1993

The glutathione S-transferase mu polymorphism as a marker for susceptibility to lung carcinoma.

Glutathione S-transferase (GST) enzymes detoxify carcinogens in tobacco smoke. Interindividual variation in GST function may be related to differences in risk for smoking-related cancer. Leukocytes from 50% of Caucasians lack GST activity toward trans-stilbene oxide (TSO), due to a deletion of the gene for the GST-mu enzyme. Presence of GST-TSO activity in leukocytes has been associated with low risk for lung cancer among cigarette smokers. We sought to determine whether GST activity in lung tissue is determined by the same gene polymorphism and whether it is associated with risk for lung cancer. Subjects were cigarette smokers, identified at the time of lung resection or autopsy in Seattle hospitals. Uninvolved lung tissue was obtained from 35 patients with lung carcinoma and 43 control patients and assayed for GST-mu activity with TSO, for the presence of the GST-mu gene product with an immunological assay, and for the GST-mu gene with Southern blotting. Mailed questionnaires were used to collect information on subjects' smoking histories and exposures which might alter enzyme activity. Interindividual results from the three assays correlated well. Smokers with high GST-TSO enzyme activity present in their lung tissue had a lower risk for lung carcinoma than did smokers with no or low activity (relative risk = 0.30; 95% confidence interval, 0.11-0.79), as did smokers with GST-mu antigen identified in lung tissue versus those with no antigen (relative risk = 0.30; 95% confidence interval, 0.11-0.79). Smokers with both maternal and paternal copies of GST-mu DNA (n = 7) had a lower cancer risk than smokers lacking GST-mu DNA (n = 30; relative risk = 0.35; 95% confidence interval, 0.06-2.10). High GST-mu activity appeared to be associated with a greater decrease in lung cancer risk among 38 heavy cigarette smokers (relative risk = 0.15; 95% confidence interval, 0.03-0.64) than among 38 light smokers (relative risk = 0.61; 95% confidence interval, 0.14-2.60). Presence or absence and number of copies of the GST-mu gene appear to determine activity of the GST-mu enzyme in lung. Smokers with the GST-mu enzyme have approximately one-third of the risk for lung carcinoma of smokers without the enzyme.

Adult↗

PubMed2002

A testing algorithm for determination of HER2 status in patients with breast cancer.

The HER2 gene is amplified and overexpressed in 25-30% of breast carcinomas. Assessment of HER2 status for prognosis and treatment of breast cancer patients can be performed by immunohistochemistry and/or fluorescence in situ hybridization (FISH). To develop a testing algorithm for HER2 in breast cancers, we used FISH analysis to determine the HER2 gene copy number and immunostaining to detect the p185 protein. Interlaboratory, interobserver, and intermethod variabilities of immunohistochemistry were assessed. In 24 invasive breast carcinomas, the indices of HER2 status obtained by FISH and by a reference laboratory's DAKO HercepTest (immunostain) gave an overall concordance of 96%. The reference laboratory's stained slides were re-interpreted by an in-house panel of pathologists; the interpretation differed in one case. The panel's interpretations were concordant with the FISH results in all 24 cases. Interobserver variability for the panel's immunohistochemistry interpretations was assessed using three different immunostaining methods on 70 slides. The numerical (0-1+, 2+, 3+) scores showed greater variability among observers than did the overall positive/negative results. One pathologist reported inconsistent results in >30% of the slides evaluated. Borderline scoring of 1-2+ was reported in 18 slides (23%) by at least one observer. Incongruent interobserver immunohistochemistry scores, leading to different positive and negative interpretations, were obtained with 5 slides (7%). The majority of consensus positive cases exhibited strong membrane staining (3+). The majority of consensus negative cases scored as 0. Based on these observations, we developed a testing algorithm that maximizes the benefits of FISH and immunohistochemistry, providing physicians with accurate results for appropriate clinical care.

Algorithms↗

PubMed1996

Interrater agreement of computed tomography infarct measurement.

To determine the reliability of infarct measurements on hard-copy computed tomography (CT) images the in vivo (IV) infarct volumes of 20 CT-detected infarcts were estimated and divided into four size groups with 5 infarcts in each group: Group A, less than 0.5 ml; Group B, 0.5 to 5.0 ml; Group C, 5 to 50 ml; and Group D, more than 50 ml. Seventeen infarcts were measured once and 3 infarcts three times to the nearest 0.5 mm by each of two neurologists and two neuroradiologists using a ruler on hard-copy CT images. The longest diameter (designated AP), the greatest diameter at right angles to AP (designated LAT), and the number of slices showing the infarct were recorded and multiplied by the hard-copy minification factor to give IV dimensions. Volume (VOL) was calculated according to a previously published method. Interrater intraclass correlation coefficients for all infarcts combined were 0.98 (AP), 0.91 (LAT), and 0.97 (VOL). Using all raters' measurements for any single infarct, the difference between the largest and the smallest measurement of AP and LAT was smallest (< 6 mm IV) for Group A and largest (< 31 mm IV) for Group D. This difference was largest relative to the dimension being measured in Groups A and B, where it reached 101% of the mean of the four raters' measurements for the AP dimension being measured in Group B, and 70% of the dimension being measured in Group A. With all raters' measurements for any single infarct, the difference between the largest and smallest measurement of VOL was smallest (< 0.5 ml) for Group A and largest (< 260 ml) for Group D. This difference was largest relative to the mean volume of the infarct being measured in Group B, where it reached 153% of the mean of the four raters' measurements for VOL and reached 115% of the mean of the four raters' measurements for VOL in Group A. The authors conclude that infarcts can be measured on hard-copy images with good interrater agreement. When infarcts with a volume smaller than 5 ml are measured, differences between raters' measurements may exceed the size of the dimensions being measured.

Cerebral Infarction↗

PubMed2005

Multiple products derived from two CCL4 loci: high incidence of a new polymorphism in HIV+ patients.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV(+) individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).

Alleles↗

PubMed2001

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens.

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.

Alleles↗

PubMed2004

Evolution of the tandem repeats in thymidylate synthase enhancer region (TSER) in primates.

The upstream regulatory region of the human thymidylate synthase gene (thymidylate synthase enhancer region, TSER) is length polymorphic, attributable to variable numbers of tandemly repeated copies of a 28-bp fragment. It has been found that TSER length polymorphism is correlated to malignancy risk. To further our understanding of the origin and evolution of TSER, this region was investigated among different primates, including hominoids, two subfamilies of the Old World monkeys (OWMs): colobines and cercopithecines, and two species of the New World monkeys (NWMs). In addition to humans, our results show that length polymorphism in TSER is also present in some primate populations, although it appears that this region is length monomorphic in many other primates. We identified three unique repeat motifs in TSER and defined them as R1, R2, and R3, respectively, starting from the 3' end. The same repeat motifs from different species are more similar to each other than different repeat motifs within same species are. Such a paraphyletic pattern suggests that divergence of the three repeat motifs predated divergence of the OWMs/hominoids and the NWMs. The most recent common ancestor (MRCA) of hominoids and the OWMs probably possessed triple repeats but now double and triple repeats are two dominant types in hominoids and the OWMs. In addition, our results show that each of the three repeat motifs may be lost independently. We have also found clues that recombination was involved in formation of tandem repeat polymorphism in TSER.

Alleles↗