Differences In Repeat Length Research Articles

FMR1 CGG Repeats: Reference Levels and Race-Ethnic Variation in Women With Normal Fertility (Study of Women's Health Across the Nation).

FMR1 premutation carriers (55-199 CGG repeats), and potentially women with high normal (35-44) or low normal (<28) CGG repeats, are at risk of premature ovarian aging. The scarcity of population data on CGG repeats <45 CGG, and variation in race-ethnicity, makes it difficult to determine true associations. DNA was analyzed for FMR1 CGG repeat lengths from 803 women (386 caucasians, 219 African Americans, 102 Japanese, and 96 Chinese) from the US-based Study of Women's Health Across the Nation (SWAN). Participants had ≥1 menses in the 3 months before enrollment, ≥1 pregnancy, no history of infertility or hormonal therapy, and menopause ≥46 years. Statistical analyses used Fisher exact tests. Among these women with normal reproductive histories, significant FMR1 repeat length differences were found across race-ethnicity for both the longer (P = .0002) and the shorter (P < .0001) alleles. The trinucleotide length variance was greater for non-Asian than Asian women (P < .0001), despite identical median values. Our data indicate that short allele lengths <25 CGG on one or both alleles are more common in non-Asian than Asian women. We confirm the minor allele in the 35 to 39 CGG range among Asians as reported previously. Only 2 (0.3%) premutation carriers were identified. These data demonstrate that FMR1 distributions do vary by race-ethnicity, even within the "normal" range. This study indicates the need to control for race-ethnicity in FMR1 ovarian aging research and provides race-ethnic population data for females separated by allele.

Biophysical Underpinnings of the Repeat Length Dependence of Polyglutamine Amyloid Formation

There are now 10 expanded CAG repeat diseases in which both disease risk and age of onset are strongly dependent on the repeat length of the polyglutamine (polyQ) sequence in the disease protein. Large, polyQ-rich inclusions in patient brains and in cell and animal models are consistent with the involvement of polyQ aggregation in the disease mechanism. This possibility is reinforced by studies showing strong repeat length dependence to the aggregation process, qualitatively mirroring the repeat length dependence of disease risk. Our understanding of the underlying biophysical principles that mediate the repeat length dependence of aggregation, however, is far from complete. A previous study of simple polyQ peptides showed that N*, the size of the critical nucleus that controls onset of aggregation, decreases from unfavorable tetramer to favorable monomer over the range Q23 to Q26. These data, however, do not explain why, for all peptides exhibiting N* ∼ 1, spontaneous aggregation rates continue to increase with increasing repeat length. Here we describe a novel kinetics analyses that maps out the nonlinear dependence with repeat length of a nucleation efficiency term that is likely related to aspects of nucleus structure. This trend accounts for why nucleus size increases to tetrameric at repeat lengths of Q23 or below. Intriguingly, both aggregation and age of onset trend with repeat length in similar ways, exhibiting large changes per added Gln at low repeat lengths and small changes per added Gln at relatively long repeat lengths. Fibril stability also increases with repeat length in a nonlinear fashion.

Pharmacokinetics of intravenous immunoglobulin in multifocal motor neuropathy

Multifocal motor neuropathy (MMN) is often responsive to treatment with intravenous immunoglobulin (IVIg), but the optimal dose and intervals of IVIg maintenance treatment have not been established. Increase in IgG concentration (ΔIgG) after IVIg infusion has recently been identified as determinant of outcome in Guillain-Barré syndrome. ΔIgG may therefore represent a potentially useful biomarker to optimise IVIg dosing in patients with MMN. The aims of this study were to determine variability of IVIg pharmacokinetics in patients with MMN in relation to treatment response, and to establish whether interindividual differences in IVIg pharmacokinetics were associated with genetic polymorphisms of the endothelial IgG receptor (FcRn) which determines IgG half-life. Twenty-three patients with MMN receiving their first IVIg treatment at a cumulative dose of 2.0 g/kg in 5 days were included. A good treatment response was defined as an increase in muscle strength of at least one Medical Research Council point in minimally two muscle groups. IgG concentrations in serum were determined at baseline, at day 1 and day 5 of the IVIg course, and 3 weeks after treatment. FcRn copy number variation and differences in repeat length of the variable number of tandem repeats in the FcRn gene were determined by quantitative PCR and Sanger sequencing. Seventeen patients (74%) had a good response to treatment. Total IgG and ΔIgG levels showed large variation between patients. Mean ΔIgG was higher in IVIg responders than in non-responders, with the largest difference on day 1 (11.1 g/L vs 4.5 g/L, p=0.06), but our study lacked power to show statistically significant differences. Genetic variation in the FcRn gene was not associated with ΔIgG levels or response to treatment. IVIg pharmacokinetics varies in patients with MMN and may be associated with clinical response.

Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study

Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are the most common known genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND). We assessed whether expansion size is associated with disease severity or phenotype. We did a cross-sectional Southern blot characterisation study (Xpansize-72) in a cohort of individuals with FTD, MND, both these diseases, or no clinical phenotype. All participants had GGGGCC repeat expansions in C9ORF72, and high quality DNA was available from one or more of the frontal cortex, cerebellum, or blood. We used Southern blotting techniques and densitometry to estimate the repeat size of the most abundant expansion species. We compared repeat sizes between different tissues using Wilcoxon rank sum and Wilcoxon signed rank tests, and between disease subgroups using Kruskal-Wallis rank sum tests. We assessed the association of repeat size with age at onset and age at collection using a Spearman's test of correlation, and assessed the association between repeat size and survival after disease onset using Cox proportional hazards regression models. We included 84 individuals with C9ORF72 expansions: 35 had FTD, 16 had FTD and MND, 30 had MND, and three had no clinical phenotype. We focused our analysis on three major tissue subgroups: frontal cortex (available from 41 patients [21 with FTD, 11 with FTD and MND, and nine with MND]), cerebellum (40 patients [20 with FTD, 12 with FTD and MND, and eight with MND]), and blood (47 patients [15 with FTD, nine with FTD and MND, and 23 with MND] and three carriers who had no clinical phenotype). Repeat lengths in the cerebellum were smaller (median 12·3 kb [about 1667 repeat units], IQR 11·1-14·3) than those in the frontal cortex (33·8 kb [about 5250 repeat units], 23·5-44·9; p<0·0001) and those in blood (18·6 kb [about 2717 repeat units], 13·9-28·1; p=0·0002). Within these tissues, we detected no difference in repeat length between disease subgroups (cerebellum p=0·96, frontal cortex p=0·27, blood p=0·10). In the frontal cortex of patients with FTD, repeat length correlated with age at onset (r=0·63; p=0·003) and age at sample collection (r=0·58; p=0·006); we did not detect such a correlation in samples from the cerebellum or blood. When assessing cerebellum samples from the overall cohort, survival after disease onset was 4·8 years (IQR 3·0-7·4) in the group with expansions greater than 1467 repeat units (the 25th percentile of repeat lengths) versus 7·4 years (6·3-10·9) in the group with smaller expansions (HR 3·27, 95% CI 1·34-7·95; p=0·009). We detected substantial variation in repeat sizes between samples from the cerebellum, frontal cortex, and blood, and longer repeat sizes in the cerebellum seem to be associated with a survival disadvantage. Our findings indicate that expansion size does affect disease severity, which--if replicated in other cohorts--could be relevant for genetic counselling. The ALS Therapy Alliance, the National Institute of Neurological Disorders and Stroke, the National Institute on Aging, the Arizona Department of Health Services, the Arizona Biomedical Research Commission, and the Michael J Fox Foundation for Parkinson's Research.

The Neurology and Corresponding Genetics of Fragile X Disorders: Insights Into the Genetics of Neurodegeneration

There have been significant advances in understanding how the fragile X gene (FMR1) can lead to distinct neurological syndromes. Clinical features of two disorders – fragile X syndrome and fragile X-associated tremor ataxia syndrome (FXTAS) – are highlighted in this article. These two disorders – one a neurodevelopmental disorder, the other a neurodegenerative disorder – are caused by a single expanded CGG repeat sequence within the FMR1 gene. Minor differences in repeat length result in the markedly different phenotypes. Understanding the action of FMR1 in FXTAS and fragile X syndrome has yielded significant insights into the genetics of neurodegeneration. Moreover, the genetic model in FXTAS is similar to several other neurologic genetic disorders, suggesting there are common pathways shared by many phenotypically diverse progressive neurodegenerative disorders. Finally, it is possible that targeted therapies for disorders such as FXTAS may also be effective in other neurodegenerative disorders that share similar mechanisms of pathogenesis.

Effects of the Pimelic Diphenylamide Histone Deacetylase Inhibitor HDACi 4b on the R6/2 and N171-82Q Mouse Models of Huntington’s Disease

This report represents a detailed description of experiments designed to replicate and extend the findings of a published study on the effects of treating the R6/2 Huntington’s disease (HD) mouse model with ~300 CAG repeats using the pimelic diphenylamide histone deacetylase (HDAC) inhibitor, HDACi 4b (Thomas et al., 2008). In addition to testing the R6/2 mice, similar experiments examined the effects of the drug on a second transgenic HD mouse model, the N171-82Q mice. As in the original study, the drug was delivered in the drinking water. In the present study we tested larger groups of mice than in the original study. The results indicated that we were unable to replicate the significant behavioral effects of oral HDACi 4b treatment in the R6/2 mice. There were however, non-significant trends for the treated R6/2 mice to be less affected on some of the measures and there were instances of phenotype progression being delayed in these treated mice. In contrast, we did replicate the protection from striatal atrophy in the R6/2 mice. We also did not observe any beneficial effects of HDACi 4b treatment in the N171-82Q mice. Although the behavioral procedures were replicated and an automated activity assessment was added, there were several unexpected complications in terms of solubility of the drug, CAG repeat length differences and gender differences in progression of the phenotype that could have affected outcomes. Clearly more studies will have to be performed using other methods of delivery as well as assessing effects in more slowly progressing HD models to better evaluate the effects of this HDAC inhibitor.

Homepeptide Repeats: Implications for Protein Structure, Function and Evolution

Analysis of protein sequences from Mycobacterium tuberculosis H37Rv (Mtb H37Rv) was performed to identify homopeptide repeat-containing proteins (HRCPs). Functional annotation of the HRCPs showed that they are preferentially involved in cellular metabolism. Furthermore, these homopeptide repeats might play some specific roles in protein–protein interaction. Repeat length differences among Bacteria, Archaea and Eukaryotes were calculated in order to identify the conservation of the repeats in these divergent kingdoms. From the results, it was evident that these repeats have a higher degree of conservation in Bacteria and Archaea than in Eukaryotes. In addition, there seems to be a direct correlation between the repeat length difference and the degree of divergence between the species. Our study supports the hypothesis that the presence of homopeptide repeats influences the rate of evolution of the protein sequences in which they are embedded. Thus, homopeptide repeat may have structural, functional and evolutionary implications on proteins.

Abstract 3048: Enhanced androgen signalling pathway increases the risk of prostate cancer through induction of TMPRSS2:ERG fusion

Abstract Clinical incidence and mortality rates of prostate cancer vary dramatically among different countries. In a recent study, we associated the difference in clinical incidence with certain somatic genomic alterations, including PTEN deletion and TMPRSS2:ERG fusion, which are found more commonly in Western but rarely in Chinese cancer samples (Mao et al, Cancer Res, 2010; 70:5207-12). TMPRSS2:ERG fusion, which is transcriptionally controlled by androgen receptor (AR), has been reported in more than 50% of prostate cancers. We investigated whether androgen level and AR activity, which varies between Chinese and Western men, could induce this fusion gene and consequently prostate cancer development. Since AR activity is inversely associated with its exon1 CAG repeats length, we determined the differences in repeat length between TMPRSS2:ERG fusion positive and negative UK samples and also between UK and China prostate cancer patients. We found significantly shorter CAG repeat lengths in the UK compared to Chinese cases (P&amp;lt;0.05). We also observed short CAG repeat lengths in the fusion positive (average n=20.3) compared to negative (average n=21.3) cases, although it is not statistically significant (p=0.14) and potentially due to the limited number of samples. We then investigated whether AR stimulation by androgen can induce TMPRSS2:ERG fusion and spatial genome reorganization. We induced the fusion not only in prostate cancer cells, but also in non-malignant prostate epithelial cells following long-term exposure to dihydrotestosterone (DHT). The induced fusion is associated with androgen-stimulated TMPRSS2 and ERG gene proximity. During our study, androgen-induced TMPRSS2 and ERG proximity and fusion were reported, but only in cancer cells (Mani et al, Science, 2009; 326:1230; Lin et al, Cell, 2009; 139:1069-83; and Haffner et al, Nat Genet, 2010; 42:668-75). We also observed that androgen-stimulated gene proximity is associated with AR transcriptional activity, which can be blocked by either RNA polymerase or AR inhibitors. Finally, we investigated the role of double strand breaks in the induction of TMPRSS2:ERG fusion. We associated high frequency of fusion induction with low expression of PIWIL1, which acts as a double strand breaks protector. Overexpression of PIWIL1 prevents DHT induced TMPRSS2:ERG fusion. Interestingly, long term DHT exposure tends to reduce expression of PIWIL1. This study demonstrates that enhanced AR activity can induce genetic alterations such as TMPRSS2:ERG fusion and consequently result in prostate carcinogenesis. The difference in physiological androgen level and AR activity may account for the variation in prostate cancer risk between Western and Chinese men and suggests that control of androgen level or AR activity can potentially prevent prostate carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3048. doi:10.1158/1538-7445.AM2011-3048

Genomic plasticity of the MHC class I A region in rhesus macaques: extensive haplotype diversity at the population level as revealed by microsatellites

The Mamu-A genes of the rhesus macaque show different degrees of polymorphism, transcription level variation, and differential haplotype distribution. Per haplotype, usually one “major” transcribed gene is present, A1 (A7), in various combinations with “minor” genes, A2 to A6. In silico analysis of the physical map of a heterozygous animal revealed the presence of similar Mamu-A regions consisting of four duplication units, but with dissimilar positions of the A1 genes on both haplotypes, and in combination with different minor genes. Two microsatellites, D6S2854 and D6S2859, have been selected as potential tools to characterize this complex region. Subsequent analysis of a large breeding colony resulted in the description of highly discriminative patterns, displaying copy number variation in concert with microsatellite repeat length differences. Sequencing and segregation analyses revealed that these patterns are unique for each Mamu-A haplotype. In animals of Indian, Burmese, and Chinese origin, 19, 15, or 9 haplotypes, respectively, could be defined, illustrating the occurrence of differential block duplications and subsequent rearrangements by recombination. The haplotypes can be assigned to 12 unique combinations of genes (region configurations). Although most configurations harbor two transcribed A genes, one or three genes per haplotype are also present. Additionally, haplotypes lacking an A1 gene or with an A1 duplication appear to exist. The presence of different transcribed A genes/alleles in monkeys from various origins may have an impact on differential disease susceptibilities. The high-throughput microsatellite technique will be a valuable tool in animal selection for diverse biomedical research projects.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-010-0486-4) contains supplementary material, which is available to authorized users.

Long tandem repeats as a form of genomic copy number variation: structure and length polymorphism of a chromosome 5p repeat in control and schizophrenia populations

Genomic copy number variations (CNVs) are a major form of variation in the human genome and play an etiologic role in several neuropsychiatric diseases. Tandem repeats, particularly with long (>50 bp) repeat units, are a relatively common yet underexplored type of CNV that may significantly contribute to human genomic variation and disease risk. We therefore carried out a pilot experiment to explore the potential role of long tandem repeats as risk factors in psychiatric disorders. A bacterial artificial chromosome-based array comparative genomic hybridization (aCGH) platform was used to examine CNVs in genomic DNA from 34 probands with schizophrenia or schizoaffective disorder. The aCGH screen detected an apparent deletion on 5p15.1 in two probands, caused by the presence in each proband of two low copy number (short) alleles of a tandem repeat that ranges in length from fewer than 10 to greater than 50 3.4 kb units in the population examined. Short alleles partially segregate with schizophrenia in a small number of families, though linkage was not significant. An association study showed no significant difference in repeat length between 406 schizophrenia cases and 392 controls. Although we did not demonstrate a relationship between the 5p15.1 repeat and schizophrenia, our results illustrate that long tandem repeats represent an intriguing type of genetic variation that have not been studied in earlier connection with psychiatric illness. aCGH can detect a small subset of these repeats, but systematic investigation will require the development of specific arrays and improved analytical methods.

Androgen receptor gene CAG repeat polymorphism in women with polycystic ovary syndrome

To evaluate the role of the androgen receptor (AR) gene CAG repeat in women with polycystic ovary syndrome (PCOS). Case control study. University department of obstetrics and gynecology. Women with (n = 114) or without (n = 205) PCOS. Peripheral blood sampling was done for DNA analysis and serum hormone measurements. CAG repeat length and serum androgen levels. No statistically significant CAG repeat length differences were found between patients and controls. We conducted a detailed analysis after dividing PCOS patients according to their free testosterone levels. The high free testosterone group had a statistically significantly longer mean biallelic average (24.0 +/- 2.0 vs. 23.0 +/- 1.5), short (22.5 +/- 1.8 vs. 21.7 +/- 1.9), and long (25.5 +/- 2.9 vs. 24.4 +/- 1.9) allelic lengths than the normal free testosterone group. In PCOS patients, a statistically significant correlation was found between biallelic average length and free testosterone concentration, either unadjusted or after adjustment. The AR gene CAG repeat polymorphism may contribute to the serum concentration of free testosterone in PCOS patients. A subset of PCOS patients with relatively longer CAG repeats (less AR activity) tended to show a higher serum androgen concentration.

How Strong Is the Association Between CAG and GGN Repeat Length Polymorphisms in the Androgen Receptor Gene and Prostate Cancer Risk?

Abstract Objective: Although narrative reviews have suggested an association between (CAG)n and (GGN)n polymorphisms in the AR gene and prostate cancer, it has never been quantified systematically. The purpose of this meta-analysis was to provide relative and absolute quantitative summary estimates with sufficient power. Method: Publications were identified through database searches for epidemiologic studies published until February 2004. For each study, mean differences in repeat length between cases and controls were calculated as well as continuous odds ratios (OR) per one CAG or GGN repeat decrement and discrete ORs to compare prostate cancer risk in men with short CAG repeats (≤21 repeats) versus long CAG repeats (&amp;gt;21 repeats) and short GGN repeats (≤16 repeats) versus long GGN repeats (&amp;gt;16 repeats). The study-specific estimates were combined by random effects metaregression analyses. Results: Nineteen case-control studies were included in this review comprising a total of 4,274 cases and 5,275 controls. Prostate cancer cases had on average 0.26 fewer CAG repeats and 0.09 fewer GGN repeats than controls. The continuous ORs of prostate cancer per one repeat decrement were 1.02 and 1.01 for CAG and GGN repeats, respectively. The summary discrete OR (95% confidence interval) were 1.19 (1.07-1.31) and 1.31 (1.06-1.61) for CAG and GGN repeat polymorphisms, respectively. Conclusion: Although the presence of shorter repeats seemed to be modestly associated with prostate cancer risk, the absolute difference in number of repeats between cases and controls is &amp;lt;1 repeat. We question whether such a small difference is enough to yield measurable biological impact in prostate carcinogenesis.

Comparative analysis of microsatellite loci in chicken and turkey

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58.

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.

Comparative analysis of microsatellite loci in chicken and turkey

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.Key words: microsatellite, chicken, turkey, Meleagris gallopavo.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

Protein misfolding is now recognized to be a central feature of neurodegenerative diseases. Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), prion diseases, and the polyglutamine (polygln) diseases all represent proteinopathies—diseases in which a particular protein or set of proteins misfolds and aggregates. In the past decade, mutations have been identified in an increasing number of genes underlying specific forms of neurodegeneration—for example, genes encoding amyloid precursor protein (APP) and presenilins 1 and 2 in AD, parkin and alpha-synuclein in PD, Cu/Zn superoxide dismutase in ALS, tau in frontotemporal dementia, prion protein in Creutzfeldt-Jacob and Gerstmann-Straussler diseases, and at least eight different proteins in the polygln diseases (Hardy and GwinnHardy 1998; Price et al. 1998). Identification of these mutations was a first step in understanding the disease process; next, it will be necessary to explain how specific changes in disease proteins may alter the neuron’s physiology and to further question how neuronal responses to the mutant protein influence the disease process. This review examines the fate of mutant protein in neurons and the potential role of posttranslational events in pathogenesis, with particular focus on the polygln diseases. Posttranslational events have already been implicated

DFLP, SSCP, and SSR markers for Δ9-stearoyl-acyl carrier protein desaturases strongly expressed in developing seeds of sunflower: intron lengths are polymorphic among elite inbred lines

Stearoyl-acyl carrier protein desaturase (SAD, EC 1.14.99.6) produces oleic acid (18:1 Δ9) by desaturating 18:0. SAD genes have been targets for breeding and engineering oilseed crops with increased stearic acid (18:0). Our aim was to clone, describe, and develop genetic markers for the SAD genes of sunflower (Helianthus annuus L.). Nineteen SAD cDNA clones were partially sequenced and found to belong to two groups. Full-length cDNAs from each group (SAD6 and SAD17) were completely sequenced. The amino acid identity of SAD6 and SAD17 was 89%. Both genes were strongly expressed in developing seeds, moderately expressed in leaves and flowers, and weakly expressed in cotyledons, roots, and stems. One intron was found in SAD6 and two introns were found in SAD17. The SAD introns from two inbred lines (HA370 and HA372) were sequenced and found to vary in length and nucleotide sequence. The length variants were caused by monomeric repeat length differences, insertions, and deletions. Three long poly-T repeats (T9 to T39) were found in one of the SAD17 introns. Three short adjacent CA repeats were found in the 5′-untranslated region of SAD6. DNA fragment length polymorphism (DFLP), single-strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for SAD6 and SAD17 by developing primers to flank introns or the CA repeats. Two of six DFLP, four of six SSCP, and one of two SSR markers were polymorphic among eight elite inbred lines. The polymorphic information contents for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Most of the polymorphisms were caused by intron fragment length polymorphisms. Introns may be an excellent source of hypervariable markers in sunflower and other crop plants.

Altered chromatin structure associated with a partial trisomy of chromosome 7 in the mouse.

The chromatin of a mouse that is trisomic for part of chromosome 7 was investigated. Chromatin from trisomic tissue has a smaller average nucleosome DNA repeat length than chromatin from tissue taken from normal diploid littermates. DNA of the nucleosome cores is the same size in both normal and trisomic tissues. Not all of the nucleosome monomers have different repeat lengths. Normal and trisomic mouse kidney cells in tissue culture maintained their nucleosome repeat-length differences.

Ribosomal RNA gene variation in diploid and tetraploid Tolmiea menziesii

Evidence from gross morphology, karyology and flavonoid chemistry suggests that Tolmiea menziesii is one of the clearest examples of autopolyploidy in natural populations. To provide additional data regarding the origin of the tetraploid cytotype of Tolmiea, both the 5S and 18S-25S ribosomal RNA genes were studied at the restriction enzyme level. Using restriction enzymes that cut once per repeat, the lengths of the 5S and 18S-25S ribosomal genes were estimated in diploids and tetraploid plants. There appear to be no consistent differences between diploids and tetraploids for the repeat length of the 18S-25S ribosomal genes. Furthermore, there is no significant repeat length heterogeneity within tetraploid plants for these genes. In addition, no differences in repeat length of the 5S genes were observed among the diploid and tetraploid plants analysed. The homogeneity observed among diploid and tetraploid plants for repeat length of the 5S and 18S-25S ribosomal genes is consistent with the hypothesis that the tetraploid cytotype is of autopolyploid origin.

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo.

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.