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Influences of progeny test programs on genetic evaluations of young sires.

Our purpose was to examine differences among AI progeny test programs in their effectiveness in identifying elite young sires for milk and protein. Data were from 6238 Holstein sires in five animal model evaluations of the USDA from January 1995 to February 1997. Bulls were required to be < or = 8 yr of age and to have > or = 10 daughters in production at the time of the evaluation. Bulls in AI programs were placed in nine groups based on affiliation with major AI organizations; from these nine groups, bulls with daughters averaging < 150 DIM and bulls sampled in > 100 herds were placed in two separate groups. Bulls sampled in organizations with controller numbers > or = 30 and bulls that had no connection to a sampling organization formed two additional groups. We also classified bulls by sampling codes of the National Association of Animal Breeders. A model predicting daughter yield deviation included effects of organization or sampling code, parent average (free of progeny information), and the interaction of organization or sampling method and parent average. When all data were used, a common intercept for milk was appropriate for all sampling methods, but the slopes differed (R2 = 0.44). Neither a common intercept nor a common slope was appropriate for protein. When data were restricted to the nine major organizations, a common intercept and slope were appropriate for milk, and R2 decreased to 0.14. A common intercept and slope were found for protein, and R2 decreased to 0.15. We detected no important differences in response to pedigree selection among progeny-testing methods used by major organizations that provide semen, but a difference was detected among the sampling codes of the National Association of Animal Breeders.

Animals↗

Screening and biochemical characterization of transthyretin variants in the Portuguese population.

The study of pathogenic and nonpathogenic transthyretin (TTR) variants is very important for the understanding of such TTR-related diseases as hereditary amyloidosis and also to establish a relationship between the structure and function of the molecule. Variants with clinical manifestations can be easily detected, but clinically silent variants can be detected only by population screening programs using specialized techniques. Hybrid isoelectric focusing (HIEF) in extremely flattened immobilized pH gradients (IPG) allows the detection of even neutral amino acid substitutions and has been used to analyze approximately 5,000 samples from the Portuguese population. Comparison with samples from carriers of three known TTR mutations (Met 30 associated with hereditary amyloidosis, Met 119, and Asn 90) was also made. In this study we detected: (1) 8 individuals carriers of TTR Met 30, (2) 35 carriers of TTR Met 119, (3) 12 carriers of TTR Asn 90, (4) 1 compound heterozygote for TTR Met 30/Met 119, and (5) 5 variants that presented a different pattern from the controls used. We also performed DNA sequencing analyses of two of the variants with the different band pattern in HIEF. The individuals were found to be carriers of TTR Ile 122 and TTR Thr 190, respectively. All the mutations detected, except for Asn 90, result from substitutions in CpG hot spots and thus can be rather frequent in the populations. Studies on the clinical evolution of the compound heterozygotes and on the physical-chemical properties of these hybrid TTRs will help to understand the pathogenicity associated with TTR.

Amyloidosis↗

Genetic instability and strain degeneration in Streptomyces rimosus.

During a strain selection program to improve oxytetracycline production in Streptomyces rimosus R6, isolates that showed extreme morphological instability appeared. Propagation via spores gave much higher instability than did propagation via mycelial fragments. Five phenotypic traits were affected: sporulation, pigmentation, colony morphology, oxytetracycline production, and oxytetracycline resistance. The variants were classified on the basis of oxytetracycline resistance into three classes. Class I variants (99% of variants) showed parental levels of resistance but were very heterogeneous regarding the other phenotypes. No DNA rearrangements were detected in primary class I variants. Class II variants (1% of variants, oxytetracycline sensitive) were phenotypically uniform, and most variants carried the same large deletion of ca. 455 kb, including the oxytetracycline resistance gene otrB. Class III variants (0.1% of variants, increased oxytetracycline resistance) were phenotypically uniform and overproduced a brown pigment and oxytetracycline. Most of these variants also showed a reproducible large-scale DNA rearrangement, which probably included deletion and a low-level reiteration (three or four copies) of a DNA fragment. "Revertants" of some class I variants show a similar DNA rearrangement to the class III variants, but there is extensive reiteration of sequences of about 200 kb, including the otrB gene. The significance of these results for the problem of strain degeneration and overproduction of antibiotics is discussed.

DNA, Bacterial↗

[Studies on the psychopathology of adults with 4-5 c/s EEG rhythm (author's transl)].

Nineteen subjects (10 males and 9 females, aged between 18 and 61 years) with an occipital 4-5 c/s EEG rhythm were tested using a multidimensional psychological test program. The program consisted of the IST of Amthauer, the Raven Test, the German version of the MMPI ("MMPI Saarbrücken"), the Cattell 16-PF-questionnaire, Brickenkamp's d2-Test, and a test battery for psychomotor impairment. A group of normal persons with a regular alpha EEG, matched according to sex and age, was used to provide test norms for the 4-5 c/s EEG group. No difference in intelligence was found, but statistically significant differences were found in some personality traits in the MMPI and the 16-PF-questionnaire. Taking into consideration that the 4-5 c/s EEG group of the study is a subjective sample not randomly selected, this study showed that the male subjects with a 4-5 c/s rhythm as more sensitive, more tender and gentle, more anxious, and less tolerant of stress when compared with the control group; in terms of sex-specific personality traits, they tend to show more female features. Furthermore, the clinical observations of the psychological test date are discussed, i.e., that the subjects with 4-5 c/s rhythms suffer more frequently from functional somatic complaints (observed in 13 cases: 9 with neurovegetative disturbances, 4 with psychosomatic diseases) and/or show psychopathological features (observed in 12 cases: 8 with abnormal personality traits, 4 with neurotic personality development).

Adolescent↗

Mutagenesis by chemical agents in V79 chinese hamster cells: a review and analysis of the literature. A report of the Gene-Tox Program.

The report reviews and evaluates the current literature (about 125 primary publications) on chemically induced specific locus mutations in the V79 Chinese hamster lung cell line. The V79 cell is convenient to use for mutagenesis studies since it has a rapid growth rate, high plating efficiency, and a stable karyotype. Mutation can be easily measured at either the hypoxanthine-guanine phosphoribosyl transferase or the Na+/K+ ATPase locus, both of which have been well characterized. Other less-studied markers are also described. We discuss the protocols for quantitative mutation studies including measurements of cytotoxicity, mutant expression times, mutant selection agents, cell densities during selection, and the stability and verification of mutant phenotypes. Mutations in the V79 cells by chemicals that require activation can be tested after their metabolism by cell homogenates or by intact cells, and the results with each type of activation are compared. For purposes of analysis, we classified a compound as mutagenic if it induced a mutation frequency that is at least 3 times higher than the spontaneous mutant frequency reported for that specific experiment. By this criterion two-thirds of the chemicals analyzed were mutagenic--; 11% with and 55% without metabolic activation. Of the 191 chemicals examined; 119 were polycyclic aromatic hydrocarbons; 25 were nitro or nitroso compounds, 9 were alkyl halides; 7 were purine or pyrimidine derivatives and the remaining 31 were from other chemical classes. We also defined mutagenic potency as the concentration of a compound that increases the mutant frequency by 10 times the spontaneous frequency. Mutagenic potencies of the compounds examined varied over a range of 5 X 10(6). We have also found large interlaboratory variations in the mutagenic potencies. Such variation in potency could be reduced by normalizing the results to a standard mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine. The role of the V79 assay in mutagenicity and carcinogenicity testing is discussed and recommendations are suggested for future investigation.

Animals↗

(Co)variance components, genetic parameters and annual trends for calf weights in a Brahman herd kept on floodable savanna.

(Co)variance components and genetic parameters were estimated for body weights of an elite Brahman herd under a designed, supervised management and genetic program, including strategic artificial insemination (AI). Restricted maximum likelihood methods were used with a univariate animal model for birth weight (BW) and a bivariate model for weaning weight (205-day weight, 205W) and 18-month weight (548-day weight, 548W). Models included random animal direct and maternal genetic effects, maternal permanent environmental effect (c2), and sex-year-month of birth-age of dam and genetic group (identified and unidentified paternity), as fixed effects. Analysis A1 included all calves and analysis A2 included only those with identified sires. Of the 8,066 calves born, 36% were progeny of AI, 11% from single sire and 53% from multi-sire herds. They were born from 1985 to 1998, from 2559 dams and 146 sires (78 identified). Estimates of direct, maternal and total heritabilities from A1 for BW, 205W and 548W were: 0.23, 0.07 and 0.30; 0.08, 0.14 and 0.16; 0.16, 0.04 and 0.28, respectively. Corresponding estimates of direct maternal genetic correlations were 0.22, 0.07 and 0.86, and c2 estimates were 0.04, 0.14 and 0.04, respectively. Estimates of direct and maternal genetic, and permanent environmental correlations between 205W and 548W were: 0.66, 0.70 and 1.00. Variances and genetic parameters from A1 and A2 were, in general, very similar. Estimates of phenotypic, and direct and maternal genetic trends per year from A1 were: 0.393, 0.004 and 0.003 kg (BW), 3.367, 0.142 and 0.115 kg (205W), 1.813, 0.263 and 0.095 kg (548W). Estimates of direct and maternal genetic trends from A2 were: 0.033 and -0.002 kg (BW); 0.186 and 0.276 kg (205W); 0.471 and 0.136 kg (548W). The modern selection methods that have been used recently should be continued, with emphasis on the improvement of cow efficiency for sustainable beef production on floodable savanna combined with improved pasture.

Analysis of Variance↗

Dual-dimensional profiling of host genomic variations and HPV integration in PD-L1-stratified cervical cancer via Oxford Nanopore Technology.

BACKGROUND: The integration of human papillomavirus (HPV) DNA into the host genome is a key step in the development of HPV-associated cervical cancer (CC). However, the genomic characteristics of host genomic variations and HPV integration within the context of programmed death-ligand 1 (PD-L1) expression stratification have not been systematically investigated. METHODS: Whole-genome sequencing was performed using Oxford Nanopore Technology (ONT) on six samples (three from the high PD-L1 expression group and three from the low PD-L1 expression group). The characteristics of host genomic variations under different PD-L1 expression stratifications were explored, including structural variations (SV), copy number variations (CNV), single nucleotide polymorphisms (SNP), and insertion-deletions (Indel). Subsequently, the distribution features of HPV integration sites were analyzed, different integration types were identified, and pathway analysis was conducted. RESULTS: Whole-genome SV analysis revealed that the total number of SVs and the composition of mutation types were similar between the high and low PD-L1 expression groups, with insertions (INS) and deletions (DEL) predominating in both. These variations were primarily enriched in intergenic regions and introns. In the low PD-L1 expression group, integration events were observed at multiple chromosomal loci, with the most frequent integration occurring in the KLF5 gene region on chromosome 13. No frequently integrated loci were identified in the high PD-L1 expression group. Additionally, four distinct HPV integration breakpoint patterns were preliminarily identified and analyzed. CONCLUSION: PD-L1 expression stratification did not significantly alter the overall genomic instability of the host. However, differences were observed in the distribution patterns of HPV integration sites. These findings provide new insights into the genomic heterogeneity of CC under different PD-L1 expression backgrounds and may lay the groundwork for future research exploring stratified immunotherapy based on HPV integration features.

Humans↗

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype.

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.

Amino Acid Sequence↗

Detection of quantitative trait loci affecting lameness and leg conformation traits in Danish Holstein cattle.

Lameness is an important factor for culling animals. Strong legs and feet improve herd life of dairy cows. Therefore, many countries include leg and feet conformation traits in their breeding programs, often as early predictors of longevity. However, few countries directly measure lameness related traits to include these in a breeding program. Lameness indices in 3 different lactations and 5 leg conformation traits (rear legs side view, rear legs rear view, hock quality, bone quality, and foot angle) were measured on granddaughters of 19 Danish Holstein grandsires with 33 to 105 sons. A genome scan was performed to detect quantitative trait loci (QTL) based on the 29 autosomes using microsatellite markers. Data were analyzed across and within families for QTL affecting lameness and leg conformation traits. A regression method and a variance component method were used for QTL detection. Two QTL each for lameness in the first [Bos taurus autosome (BTA); BTA5, BTA26] and second (BTA19, BTA22) lactations were detected. For the 5 different leg conformation traits, 7 chromosome-wise significant QTL were detected across families for rear legs side view, 5 for rear legs rear view, 4 for hock quality, 4 for bone quality, and 1 for foot angle. For those chromosomes where a QTL associated with 2 different traits was detected (BTA1, BTA11, BTA15, BTA26, and BTA27), a multitrait-1-QTL model and a multitrait-2-QTL model were performed to characterize these QTL as single QTL with pleiotropic effects or distinct QTL.

Animals↗

Effects of genotype x environment interaction on genetic gain in breeding programs.

Genotype x environment interaction (G x E) is increasingly important, because breeding programs tend to be more internationally oriented. The aim of this theoretical study was to investigate the effects of G x E on genetic gain in sib-testing and progeny-testing schemes. Loss of genetic gain due to G x E was predicted for different values of heritability, number of progeny per dam, number of progeny per sire, proportion of selected sires, and population size in the selection environment. Two environments were considered: a selection environment (SLE) and a production environment (PDE). The breeding goal was only for performance in PDE. A pseudo-BLUP selection index was used to predict genetic gain. Recording of half-sibs or progeny in PDE limited the loss in genetic gain in PDE due to G x E between SLE and PDE. Progeny-testing schemes had less loss in genetic gain than sib-testing schemes. Higher heritability increased the loss in genetic gain, whereas increasing the number of progeny per sire in PDE decreased the loss in genetic gain. The number of progeny per sire required to minimize loss in genetic gain due to G x E was greater for sib-testing schemes than for progeny-testing schemes. More progeny per dam slightly increased the loss in genetic gain. Genetic gains for sex-limited and carcass traits were less affected by G x E than traits measured on both sexes. Loss in genetic gain was due to decreased accuracy of selection in most situations, but it was due to decreased selection intensity in situations with small population size and a low proportion of selected sires. It was concluded that recording performance of relatives in PDE minimizes loss in genetic gain due to G x E, and that progeny-testing schemes rather than sib-testing schemes are preferable in situations with low to moderate heritability (h(2) <or= 0.3), relatively short generation interval of progeny-tested sires (L(prog)/L(sib) <or= 1.7), and moderate to severe G x E interaction (r(g) <or= 0.8).

Animals↗

In vitro fertilization: a cost-effective alternative for infertile couples?

OBJECTIVE: To evaluate the cost of in vitro fertilization by calculating the cost of a live birth using this technology and determine cost variation according to the clinical characteristics of a particular population. DESIGN: Retrospective review of infertile couples who presented for their first IVF cycle in 1993. A fraction of the total population was assigned to three groups A, B, and C with high, intermediate and low probability of pregnancy respectively and their reproductive performance was evaluated until September 1994 or a maximum of three IVF cycles have been completed. SETTING: The in vitro fertilization program at the Brigham and Women's Hospital, Boston. PATIENTS: 182 couples who presented for their first IVF cycle in 1993. MAIN OUTCOME MEASURE: The cost of a successful pregnancy using IVF in the three groups and in the general population was calculated by dividing the average cost of an IVF cycle by the fraction of the cycles resulting in a successful pregnancy. RESULTS: The cost of a successful pregnancy in group A, B and C ranged from $22,857 to $42,666 after 1 cycle and from $26,800 to $74,666 after 3 IVF cycles. The average cost for the 182 patients was $29,120 after 1 cycle and $31,590 after a maximum of 3 IVF cycles. CONCLUSION: The cost of a successful pregnancy: (1) was comparable to other options available to an infertile couple such as adoption and tubal surgery, (2) was 50% to 70% cheaper in the group with a highest probability of pregnancy when compared to the group with the lowest probability of pregnancy, and (3) did not vary significantly after 1 or 3 IVF cycles in most groups.

Cost-Benefit Analysis↗

Irregular polymerase chain reaction-sequence-specific oligonucleotide hybridization patterns reveal seven new HLA-DRB1 alleles related to DR2, DR3, DR6, DR8, and DR11. Implications for sequence-specific priming.

In the past 3 years we have typed over 7000 individuals for HLA-DRB using a nonradioactive PCR-SSO method. The use of locally developed computer programs simplified data input and the interpretation of the DRB PCR-SSO readings. In this way we detected a number of samples with unexpected hybridization patterns. DRB1 exon 2 segments of these samples were amplified, cloned, and sequenced and appeared to identify seven new DRB alleles: DRB1*0304, a DRB1*0301 variant, was observed in three unrelated Caucasoid individuals; DRB1*1606, which is very similar to *1603; DRB1*1113 is a *1101 variant with some *1401 sequences; DRB1*1310 is *1301-like; DRB1*1311 is similar to *1305 and *1307; DRB1*1416 is a *1401 sequence with a HV3 derived from *1301; DRB1*0808 was found in an Ethiopian individual. Next, we studied the effectiveness of PCR-SSP typing of the newly defined DRB1 alleles. Only two variants were distinguished as odd by PCR-SSP and two were typed as regular specificities. Three alleles were not amplified by the primer sets used. As compared to PCR-SSO, the PCR-SSP typing method using currently available typing kits clearly has limitations as far as the recognition of new and variant alleles is concerned. The products of some of these new alleles may be distinguished using conventional serology.

Alleles↗

Gene expression in single cells of Bacillus subtilis: evidence that a threshold mechanism controls the initiation of sporulation.

Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis↗

Organic aciduria and butyryl CoA dehydrogenase deficiency in BALB/cByJ mice.

A metabolic screening program of inbred strains of mice has detected a marked organic aciduria in the BALB/cByJ strain. Gas chromatographic and mass spectrometric analysis identified large quantities of n-butyrylglycine plus lesser quantities of ethylmalonic acid. Crosses with the nonexcreting C57BL/6J strain indicate that this condition is inherited as an autosomal recessive trait. Independently from this screening a variant with no detectable enzyme activity of butyryl CoA dehydrogenase (BCD) in liver and kidney of the BALB/cByJ strain but not other BALB/c sublines was discovered. Data from a three-point cross indicated that the null variant maps to the structural locus for the enzyme, Bcd-1, on chromosome 5. The findings indicate that a mutation at or near Bcd-1 in the BALB/cByJ strain resulted in a biochemical abnormality manifest as the BCD deficiency. It is concluded that accumulation of butyryl CoA due to a block in the oxidation of short-chain fatty acids results in an overproduction of organic metabolites leading to the observed organic aciduria. The fact that other BALB/c substrains do not exhibit this abnormality further suggests that this disorder reflects subline divergence within the BALB/c family.

Animals↗

CGHScan: finding variable regions using high-density microarray comparative genomic hybridization data.

BACKGROUND: Comparative genomic hybridization can rapidly identify chromosomal regions that vary between organisms and tissues. This technique has been applied to detecting differences between normal and cancerous tissues in eukaryotes as well as genomic variability in microbial strains and species. The density of oligonucleotide probes available on current microarray platforms is particularly well-suited for comparisons of organisms with smaller genomes like bacteria and yeast where an entire genome can be assayed on a single microarray with high resolution. Available methods for analyzing these experiments typically confine analyses to data from pre-defined annotated genome features, such as entire genes. Many of these methods are ill suited for datasets with the number of measurements typical of high-density microarrays. RESULTS: We present an algorithm for analyzing microarray hybridization data to aid identification of regions that vary between an unsequenced genome and a sequenced reference genome. The program, CGHScan, uses an iterative random walk approach integrating multi-layered significance testing to detect these regions from comparative genomic hybridization data. The algorithm tolerates a high level of noise in measurements of individual probe intensities and is relatively insensitive to the choice of method for normalizing probe intensity values and identifying probes that differ between samples. When applied to comparative genomic hybridization data from a published experiment, CGHScan identified eight of nine known deletions in a Brucella ovis strain as compared to Brucella melitensis. The same result was obtained using two different normalization methods and two different scores to classify data for individual probes as representing conserved or variable genomic regions. The undetected region is a small (58 base pair) deletion that is below the resolution of CGHScan given the array design employed in the study. CONCLUSION: CGHScan is an effective tool for analyzing comparative genomic hybridization data from high-density microarrays. The algorithm is capable of accurately identifying known variable regions and is tolerant of high noise and varying methods of data preprocessing. Statistical analysis is used to define each variable region providing a robust and reliable method for rapid identification of genomic differences independent of annotated gene boundaries.

Algorithms↗

Efficiency of a programmed -1 ribosomal frameshift in the different subtypes of the human immunodeficiency virus type 1 group M.

The synthesis of the Gag-Pol polyprotein, the precursor of the enzymes of the human immunodeficiency virus type 1 (HIV-1), requires a programmed -1 ribosomal frameshift. This frameshift has been investigated so far only for subtype B of HIV-1 group M. In this subtype, the frameshift stimulatory signal was found to be a two-stem helix, in which a three-purine bulge interrupts the two stems. In this study, using a luciferase reporter system, we compare, for the first time, the frameshift efficiency of all the subtypes of group M. Mutants of subtype B, including a natural variant were also investigated. Our results with mutants of subtype B confirm that the bulge and the lower stem of the frameshift stimulatory signal contribute to the frameshift in addition to the upper stem-loop considered previously as the sole participant. Our results also show that the frameshift stimulatory signal of all of the other subtypes of group M can be folded into the same structure as in subtype B, despite sequence variations. Moreover, the frameshift efficiency of these subtypes, when assessed in cultured cells, falls within a narrow window (the maximal deviation from the mean value calculated from the experimental values of all the subtypes being approximately 35%), although the predicted thermodynamic stability of the frameshift stimulatory signal differs between the subtypes (from -17.2 kcal/mole to -26.2 kcal/mole). The fact that the frameshift efficiencies fall within a narrow range for all of the subtypes of HIV-1 group M stresses the potential of the frameshift event as an antiviral target.

Base Sequence↗

Identification of alternatively spliced mRNA variants related to cancers by genome-wide ESTs alignment.

Several databases have been published to predict alternative splicing of mRNAs by analysing the exon linkage relationship by alignment of expressed sequence tags (ESTs) to the genome sequence; however, little effort has been made to investigate the relationship between cancers and alternative splicing. We developed a program, Alternative Splicing Assembler (ASA), to look for splicing variants of human gene transcripts by genome-wide ESTs alignment. Using ASA, we constructed the biosino alternative splicing database (BASD), which predicted splicing variants for reference sequences from the reference sequence database (RefSeq) and presented them in both graph and text formats. EST clusters that differ from the reference sequences in at least one splicing site were counted as splicing variants. Of 4322 genes screened, 3490 (81%) were observed with at least one alternative splicing variants. To discover the variants associated with cancers, tissue sources of EST sequences were extracted from the UniLib database and ESTs from the same tissue type were counted. These were regarded as the indicators for gene expression level. Using Fisher's exact test, alternative splicing variants, of which EST counts were significantly different between cancer tissues and their counterpart normal tissues, were identified. It was predicted that 2149 variants, or 383 variants after Bonferroni correction, of 26 812 variants were likely tumor-associated. By reverse transcription-PCR, 11 of 13 novel alternative splicing variants and eight of nine variants' tissue specificity were confirmed in hepatocellular carcinoma and in lung cancer. The possible involvement of alternative splicing in cancer is discussed.

Alternative Splicing↗

The solution conformations of a mutant trp operator determined by n.m.r. spectroscopy.

The principal conformational features of the mutant trp operator [d(CGTACTGATTAATCAGTACG)2] have been determined by n.m.r. at different temperatures. The sugar puckers were determined from J-resolved spectroscopy and high-resolution homonuclear Hartmann-Hahn spectroscopic (HOHAHA) experiments. Extensive one-dimensional nuclear-Overhauser-enhancement (NOE) data sets were acquired at 25 degrees C using irradiation times of 50, 100, 200, 300 and 500 ms to generate sufficient NOE information to determine the individual nucleotide conformations, and place limits on the local helical parameters, using multi-spin least-squares fitting and searching in conformation space with the program NUCFIT [Lane (1990) Biochim. Biophys. Acta 1049, 189-204]. The conformations of the nucleotides are well determined, and show significant sequence-dependent variation. Pyrimidine residues on average have a wider range of sugar conformations and smaller glycosidic torsion angles than purine residues. The helical parameters are in general less well determined, though clear evidence was obtained for sequence-dependent variation of the helical twist. The overall mean fractional deviation of the calculated from the observed NOEs was 0.108. The conformations of the base-pairs TAAT are temperature-dependent [Lane (1989) Biochem. J. 259, 715-724]. NOESY spectra were recorded at 10, 25 and 40 degrees C, using mixing times inversely proportional to the overall tumbling time to allow changes in the conformation to be described. A more detailed analysis was made using one-dimensional NOEs collected for nucleotides involved in the conformational transitions. There are significant temperature-dependent changes in the conformations of the central base-pairs from T9 to T13 with the largest changes in the glycosidic torsion angle occurring for A11 and A12 (up to 30 degrees). The orientation of the base-pairs T9-A12:T10-A11 also changes, with an increase in the base-pair roll and an unwinding of the helix as the temperature is increased. The conformational changes are qualitatively similar to those observed in a related sequence (Lefèvre, Lane & Jardetzky (1988) Biochemistry 27, 1086-1094]. The conformation is also similar to the wild-type sequence and to that observed in the crystal state in the complex with the trp holorepressor. The similarity suggests that the mutation produces a poorer operator by virtue of removal of essential functional groups within the major groove.

Base Composition↗