Unstable DNA Sequences Research Articles

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Interstitial deletions of chromosome 1p: novel 1p31.3p22.2 microdeletion in a newborn with craniosynostosis, coloboma and cleft palate, and review of the genomic and phenotypic profiles

BackgroundRearrangements of unstable DNA sequences may alter the structural integrity or the copy number of dose-sensitive genes, resulting in copy number variations. They may lead more frequently to deletions, in addition to duplications and/or inversions, which are the underlying pathogenic mechanism of a group of conditions known as genomic disorders (or also contiguous gene syndromes). Interstitial deletions of the short arm of chromosome 1 are rare, and only about 30 patients have been reported. Their clinical features are variable, in respect of the extent of the deleted region. They include global developmental delay, central nervous system (CNS) malformations, craniosynostosis, dysmorphic face, ocular defects, cleft palate, urinary tract anomalies and hand/foot abnormalities.Case presentationHereby, we report on an Italian female newborn with craniosynostosis, facial dysmorphisms including bilateral microphthalmia and coloboma, cleft palate, and a severe global developmental and growth delay, associated to a 1p31.3p22.2 deletion of 20.7 Mb. This was inherited from the healthy mother, who was carrier of a smaller (2.6 Mb) deletion included within the centromeric region (1p22.3p22.2) of the same rearrangement, in addition to a translocation between chromosomes 1p and 4q. The deleted region of the proband contains about ninety genes. We focus on the genotype–phenotype correlations.ConclusionsThe results of the present study further confirm that microdeletions at 1p31.3 constitute a contiguous gene syndrome. It is hard to establish whether the critical rearrangement of such syndrome may involve the centromeric band p22.3p22.2, or more likely do not, also in light of the genomic profile of the healthy mother of our patient. Neonatologists and pediatricians should take into consideration 1p31 microdeletion in cases of developmental and growth delay associated to craniosynostosis, peculiar facial dysmorphisms, cleft palate and hand/foot abnormalities. The present report provides new data about 1p31 microdeletion syndrome, in view of a better characterization of its genomic and phenotypic profile.

NOREC4DNA: using near-optimal rateless erasure codes for DNA storage

BackgroundDNA is a promising storage medium for high-density long-term digital data storage. Since DNA synthesis and sequencing are still relatively expensive tasks, the coding methods used to store digital data in DNA should correct errors and avoid unstable or error-prone DNA sequences. Near-optimal rateless erasure codes, also called fountain codes, are particularly interesting codes to realize high-capacity and low-error DNA storage systems, as shown by Erlich and Zielinski in their approach based on the Luby transform (LT) code. Since LT is the most basic fountain code, there is a large untapped potential for improvement in using near-optimal erasure codes for DNA storage.ResultsWe present NOREC4DNA, a software framework to use, test, compare, and improve near-optimal rateless erasure codes (NORECs) for DNA storage systems. These codes can effectively be used to store digital information in DNA and cope with the restrictions of the DNA medium. Additionally, they can adapt to possible variable lengths of DNA strands and have nearly zero overhead. We describe the design and implementation of NOREC4DNA. Furthermore, we present experimental results demonstrating that NOREC4DNA can flexibly be used to evaluate the use of NORECs in DNA storage systems. In particular, we show that NORECs that apparently have not yet been used for DNA storage, such as Raptor and Online codes, can achieve significant improvements over LT codes that were used in previous work. NOREC4DNA is available on https://github.com/umr-ds/NOREC4DNA.ConclusionNOREC4DNA is a flexible and extensible software framework for using, evaluating, and comparing NORECs for DNA storage systems.

Ethidium Bromide Modifies The Agarose Electrophoretic Mobility of CAG•CTG Alternative DNA Structures Generated by PCR.

The abnormal expansion of unstable simple sequence DNA repeats can cause human disease through a variety of mechanisms, including gene loss-of-function, toxic gain-of-function of the encoded protein and toxicity of the repeat-containing RNA transcript. Disease-associated unstable DNA repeats display unusual biophysical properties, including the ability to adopt non-B-DNA structures. CAG•CTG trinucleotide sequences, in particular, have been most extensively studied and they can fold into slipped-stranded DNA structures, which have been proposed as mutation intermediates in repeat size expansion. Here, we describe a simple assay to detect unusual DNA structures generated by PCR amplification, based on their slow electrophoretic migration in agarose and on the effects of ethidium bromide on the mobility of structural isoforms through agarose gels. Notably, the inclusion of ethidium bromide in agarose gels and running buffer eliminates the detection of additional slow-migrating DNA species, which are detected in the absence of the intercalating dye and may be incorrectly classified as mutant alleles with larger than actual expansion sizes. Denaturing and re-annealing experiments confirmed the slipped-stranded nature of the additional DNA species observed in agarose gels. Thus, we have shown that genuine non-B-DNA conformations are generated during standard PCR amplification of CAG•CTG sequences and detected by agarose gel electrophoresis. In contrast, ethidium bromide does not change the multi-band electrophoretic profiles of repeat-containing PCR products through native polyacrylamide gels. These data have implications for the analysis of trinucleotide repeat DNA and possibly other types of unstable repetitive DNA sequences by standard agarose gel electrophoresis in diagnostic and research protocols. We suggest that proper sizing of CAG•CTG PCR products in agarose gels should be performed in the presence of ethidium bromide.

DNA repair in the trinucleotide repeat disorders

Inherited diseases caused by unstable repeated DNA sequences are rare, but together represent a substantial cause of morbidity. Trinucleotide repeat disorders are severe, usually life-shortening, neurological disorders caused by nucleotide expansions, and most have no disease-modifying treatments. Longer repeat expansions are associated with genetic anticipation (ie, earlier disease onset in successive generations), although the differences in age at onset are not entirely accounted for by repeat length. Such phenotypic variation within disorders implies the existence of additional modifying factors in pathways that can potentially be modulated to treat disease. A genome-wide association study detected genetic modifiers of age at onset in Huntington's disease. Similar findings were seen in the spinocerebellar ataxias, indicating an association between DNA damage-response and repair pathways and the age at onset of disease. These studies also suggest that a common genetic mechanism modulates age at onset across polyglutamine diseases and could extend to other repeat expansion disorders. Genetic defects in DNA repair underlie other neurodegenerative disorders (eg, ataxia-telangiectasia), and DNA double-strand breaks are crucial to the modulation of early gene expression, which provides a mechanistic link between DNA repair and neurodegeneration. Mismatch and base-excision repair are important in the somatic expansion of repeated sequences in mouse models of trinucleotide repeat disorders, and somatic expansion of the expanded CAG tract in HTT correlates with age at onset of Huntington's disease and other trinucleotide repeat disorders. WHERE NEXT?: To understand the common genetic architecture of trinucleotide repeat disorders and any further genetic susceptibilities in individual disorders, genetic analysis with increased numbers of variants and sample sizes is needed, followed by sequencing approaches to define the phenotype-modifying variants. The findings must then be translated into cell biology analyses to elucidate the mechanisms through which the genetic variants operate. Genes that have roles in the DNA damage response could underpin a common DNA repeat-based mechanism and provide new therapeutic targets (and hence therapeutics) in multiple trinucleotide repeat disorders.

Paternal lifestyle as a potential source of germline mutations transmitted to offspring

Paternal exposure to high levels of radioactivity causes heritable germline minisatellite mutations. However, the effect of more general paternal exposures, such as cigarette smoking, on germline mutations remains unexplored. We analyzed two of the most commonly used minisatellite loci (CEB1 and B6.7) to identify germline mutations in blood samples of complete mother-father-child triads from the Norwegian Mother and Child Cohort Study (MoBa). The presence of mutations was subsequently related to general lifestyle factors, including paternal smoking before the partner became pregnant. Paternally derived mutations at the B6.7 locus (mutation frequency 0.07) were not affected by lifestyle. In contrast, high gross yearly income as a general measure of a healthy lifestyle coincided with low-mutation frequencies at the CEB1 locus (P=0.047). Income was inversely related to smoking behavior, and paternally derived CEB1 mutations were dose dependently increased when the father smoked in the 6 mo before pregnancy, 0.21 vs. 0.05 in smoking and nonsmoking fathers, respectively (P=0.061). These results suggest that paternal lifestyle can affect the chance of heritable mutations in unstable repetitive DNA sequences. To our knowledge, this is the first study reporting an effect of lifestyle on germline minisatellite mutation frequencies in a human population with moderate paternal exposures.

Genome hyperevolution and the success of a parasite

The strategy of antigenic variation is to present a constantly changing population phenotype that enhances parasite transmission, through evasion of immunity arising within, or existing between, host animals. Trypanosome antigenic variation occurs through spontaneous switching among members of a silent archive of many hundreds of variant surface glycoprotein (VSG) antigen genes. As with such contingency systems in other pathogens, switching appears to be triggered through inherently unstable DNA sequences. The archive occupies subtelomeres, a genome partition that promotes hypermutagenesis and, through telomere position effects, singular expression of VSG. Trypanosome antigenic variation is augmented greatly by the formation of mosaic genes from segments of pseudo-VSG, an example of implicit genetic information. Hypermutation occurs apparently evenly across the whole archive, without direct selection on individual VSG, demonstrating second-order selection of the underlying mechanisms. Coordination of antigenic variation, and thereby transmission, occurs through networking of trypanosome traits expressed at different scales from molecules to host populations.

An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

BackgroundTagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.ResultsThe Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei.ConclusionWe have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.

A modified low copy number binary vector pUN for Agrobacterium-mediated plant transformation

A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.

Cloning of genetically tagged chromosome break sequences reveals new fragile sites at 6p21 and 13q22

Fragile sites are specific genomic loci that are especially prone to chromosome breakage. For the human genome there are 31 rare fragile sites and 88 common fragile sites listed in the National Center for Biotechnology Information database; however, the exact number remains unknown. In this study, unstable DNA sequences, which have been previously tagged with a marker gene, were cloned and provided starting points for the characterization of two aphidicolin inducible common fragile sites. Mapping of these unstable regions with six-color fluorescence in situ hybridization revealed two new fragile sites at 6p21 and 13q22, which encompass genomic regions of 9.3 and 3.1 Mb, respectively. According to the fragile site nomenclature they were consequently entitled as FRA6H and FRA13E. Both identified regions are known to be associated with recurrent aberrations in malignant and nonmalignant disorders. It is conceivable that these fragile sites result in genetic damage that might contribute to cancer phenotypes such as osteosarcoma, breast and prostate cancer.

Common fragile site FRA11G and rare fragile site FRA11B at 11q23.3 encompass distinct genomic regions

Fragile sites are specific genomic loci that are particularly prone to chromosomal breakage. Based on their incidence in the human population, they are divided into rare fragile sites occurring in less than 5% of all individuals and common fragile sites being a constitutional feature of the genome of probably all individuals. In this study, cloning of unstable DNA sequences, which have been previously genetically tagged with a marker gene, was the basis for defining the genomic localization of the common fragile site FRA11G at 11q23.3. Mapping of the fragile site with six-color fluorescence in situ hybridization (FISH) resulted in the precise genomic localization of FRA11G to a 4.5 Mb region. The chromosomal subband 11q23.3 harbors both the common fragile site FRA11G and the rare fragile site FRA11B. Here, we show that FRA11G maps 0.8 Mb proximal to the genomic region previously defined to be affected by expression of FRA11B; thus, the common and the rare fragile sites at 11q23.3 encompass distinct genomic regions. The region of FRA11G is known to be involved in somatic and germline recurrent aberrations, and it is conceivable that genetic damage resulting from this fragile site might contribute to clinical phenotypes.

Non-clonability correlates with genomic instability: a case study of a unique DNA region

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the π and αD chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG∗CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of “unclonable” DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.

Anticipation in bipolar affective disorder: is age at onset a valid criterion?

Anticipation has been suggested among the genetic mechanisms of bipolar disorder (BD), prompting the search for unstable DNA sequences. Past studies of anticipation in BD have generally relied on observed shift in the age at onset between parental and offspring generations. Such a shift, however, may be caused by a number of other factors difficult to correct for. We investigated age at onset distributions in a sample of 161 related subjects and in a sample of "pseudofamilies" consisting of 320 unrelated subjects selected from a large epidemiological cohort using Monte-Carlo simulation to mimic the family sample. Comparison of age at onset distributions in both samples shows a difference between the generations, but of a similar magnitude in each sample. This suggests that age at onset alone may not be a sufficient criterion of anticipation. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:804-807, 2000.

Bipolar Disorders: Clinical Course and Outcome

As part of the Clinical Practice Series of the American Psychiatric Press, this book on bipolar disorders integrates current clinical research findings with clinical applications and provides a broad overview of course and outcome for bipolar disorders. The book provides a concise, up-to-date summary of current knowledge about affective relapse, comorbid psychopathology, functional disability, and psychosocial outcome in bipolar disorders. It has been generally acknowledged that bipolar affective disorders are one of the leading causes of chronic disability worldwide. From the perspective of illness-related cost, the total economic impact of bipolar disorder is estimated at several billion dollars. In recent decades, the diagnostic trends in the United States have favored a broadened definition of bipolar illness. Another change in diagnostic approaches may involve new phenotypes of the disorder. These phenotypes may be reflected in cohort effects among contemporary bipolar patients involving factors such as drug and alcohol abuse, past treatment with tricyclic or other antidepressant agents, and cross-generational shifts due to transmission of unstable DNA sequences. As Dr. Frederick Goodwin notes in his foreword, “One fascinating hypothesis suggests that a cross-generational shift to more malignant forms of the illness may reflect a genetic mechanism involving unstable DNA. Trinucleotide repeats would increase the severity of the illness in succeeding generations, perhaps contributing to greater treatment resistance.” Indeed, this hypothesis may explain what clinicians have known for the past several years—that bipolar patients do not appear to be responding to lithium carbonate as before. There is clearly a disparity between what is observed in clinical practice and in controlled clinical trials with lithium carbonate. Clinicians have long known that bipolar disorder is a recurrent disorder. The episodes recur, and recurrences tend to come closer together as the disorder progresses. Furthermore, the degree of environmental stress associated with recurrent episodes becomes progressively less intense over time. These phenomena have generated the “kindling hypothesis.” Another hypothesis posits that the episodic nature of the illness reflects a disturbance in the regulation of biological rhythms. These hypotheses have clinical implications. Antiseizure medications have gradually replaced lithium carbonate as the treatment of first choice for most bipolar patients. It is believed that many of these drugs, such as valproic acid, may act through biological mechanisms that attenuate the kindling phenomenon. Psychosocial factors, at least initially, play an important role in precipitating episodes of either depression or mania. In what is perhaps the best chapter, Miklowitz and Frank explore psychotherapeutic strategies for bipolar disorder. They modify a model proposed by Goodwin and Jamison that posits a role for stressful life events. The authors suggest various stages in family-focused treatment of bipolar disorder that include, after the initial assessment phase, education about bipolar disorder, communication enhancement, and problem-solving training. And based on the observation that the episodic nature of the illness reflects a disturbance in the regulation of biological rhythms, they propose strategies for interpersonal and “social rhythm therapy” of bipolar disorder. In this model, the patients are taught to regulate social rhythm and sleep/wake cycles and to understand and renegotiate the interpersonal context associated with the onset of mood disorder symptoms. Lastly, the patients learn to master conflicts associated with interpersonal loss, deficits, and role transitions. In short, this book will serve as an excellent handbook for clinicians who work with bipolar patients. The book is of value not just to psychiatrists but also to primary care physicians who very often are the first to diagnose bipolar disorder and treat the condition through various stages of the disease. I highly recommend this book.

Refining a proximal breakpoint cluster at chromosome 3p11.2 in non-papillary renal cell carcinomas.

Our previous cytogenetic and microsatellite analyses showed a terminal deletion of chromosome 3p segments with a breakpoint region between 3p11.2 and 3p14.1 bands in 98% of non-papillary renal cell carcinomas (RCC). This breakpoint region covers approx. 20-25 cM genetic distance. Earlier, we found a higher frequency of rearrangements at chromosome 3p11.2 and have therefore now analyzed 74 sporadic and 26 VHL-associated RCCs with 6 polymorphic microsatellite markers mapped to this region. Thirty-three per cent of the breakpoints were mapped to a genetic distance of less than one-twentieth of the large breakpoint region 3p11.2-14.1. We suggest that unstable DNA sequences at chromosome 3p11.2 serve as a genetic basis for deletions and homologous and non-homologous recombinations to remove distal 3p sequences with one copy of RCC gene(s) in different types of tumors. We have identified a YAC clone 909d5 that spans the most frequently occurring breaks between loci D3S1251, D3S1101, and D3S1552 and allows this region to be cloned.

The brain hallucinates and gets caught in the web

In the past year, we had a remarkable glimpse of brain activity during hallucination in a patient with schizophrenia (Silbersweig; figure). In his typical hallucination, a patient saw moving, coloured scenes with rolling, disembodied heads, which were giving him instructions. 150 positron emission tomography and an image-analysis protocol, designed to image transient subjective experiences, illustrated which areas had increased regional cerebral blood flow during the hallucinations. Increased brain activity was noted in visual and auditory-associated cortices, as well as paralimbic and subcortical areas. Such studies provide clues to the pathophysiology of schizophrenia and to the neural substrate of conscious perceptual experience. Fascinating new clues to schizophrenia also emerged on the genetics/epidemiology front. Gorwood and colleagues tested the hypothesis that the age of onset of schizophrenia decreases in successive generations. This inheritance pattern, anticipation, results from unstable trinucleotide repeat DNA sequences at the disease locus. The study took place on an island in the Indian Ocean and was based on 97 patients with schizophrenia belonging to 24 families with at least two generations of patients. In the younger generation, the age of onset was significantly less than the expected age of onset based on the data from the older generation. This evidence could accelerate the search for pathological genetic processes implicated in schizophrenia. Psychiatrists are concerned about the potential iatrogenic effects of typical antipsychotics, which are linked to tardive dyskinesia. These effects may be relieved by the findings of McCreadie and colleagues. Dyskinesia was assessed in four groups of elderly individuals including normal people, relatives of schizophrenics, nevermedicated patients with schizophrenia, and medicated patients. The prevalence of dyskinesia was similar in never-medicated and medicated patients (about 40%), and was significantly higher than that in first-degree relatives and normal controls (15%). McCreadie reported that never-medicated patients had classic tardive dysthinesia, including chewing movements, tongue protrusion, lip smacking, and writhing. The researchers conclude that it is the illness per se that is associated with dyskinesias in elderly schizophrenic patients. However, psychiatrists may be chagrined by the work of Mojtabai and Nicholson. These researchers evaluated the inter-rater reliability of ratings of delusions and bizarre hallucinations among 50 psychiatrists selected randomly from the American Psychiatry Association Membership Directory. The inter-rater reliability estimates were quite low (K about 0-40), and call into question the adequacy of psychiatric training.

Anticipation of autosomal dominant progressive external ophthalmoplegia with hypogonadism.

A large Swedish family with members affected by progressive external ophthalmoplegia with hypogonadism were followed-up and reviewed. Hypogonadism included delayed sexual maturation, primary amenorrhea, early menopause, and testicular atrophy. Cataracts, cerebellar ataxia, neuropathy, hypoacusia, pes cavus, tremor, parkinsonism, depression, and mental retardation were other features observed in this family. Muscle biopsy samples of advanced cases showed ragged-red fibers, focal cytochrome c oxidase deficiency, and multiple mtDNA deletions by Southern blot analysis. An autosomal dominant mode of inheritance was evident with anticipation in successive generations. Linkage analysis excluded the chromosome 10q23.3-q24.3 region reported as being linked to the disease in a Finnish family with autosomal dominant progressive external ophthalmoplegia. We report for the first time clinical evidence for anticipation in a family with autosomal dominant progressive external ophthalmoplegia. We hypothesize that the nuclear gene causing this enigmatic disorder may be directly influenced by an expansion of an unstable DNA sequence and that the resulting phenotype is caused by a concerted action with multiple deletions of mtDNA.

Detailed Comparative Map of Human Chromosome 19q and Related Regions of the Mouse Genome

One of the larger contiguous blocks of mouse–human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent “transpositions” of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and human regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3–q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse–human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species.

Analysis of unstable DNA sequence in FMR1 gene in Polish families with fragile X syndrome.

The unstable DNA sequence in the FMR1 gene was analyzed in 85 individuals from Polish families with fragile X syndrome in order to characterize mutations responsible for the disease in Poland. In all affected individuals classified on the basis of clinical features and expression of the fragile site at X(q27.3) a large expansion of the unstable sequence (full mutation) was detected. About 5% (2 of 43) of individuals with full mutation did not express the fragile site. Among normal alleles, ranging in size from 20 to 41 CGG repeats, allele with 29 repeats was the most frequent (37%). Transmission of premutated and fully mutated alleles to the offspring was always associated with size increase. No change in repeat number was found when normal alleles were transmitted.

Molecular structure of the transposable element ninja in Drosophila simulans.

Genetically unstable DNA sequences of 16.1 kb in length were isolated from the white locus of the W(mky) strain of Drosophila simulans. This insertional DNA has some unique characteristics as a transposon. It is found in high numbers in this strain and its revertant strains W(psm1) and W(cho), but not elsewhere, and the sequence is a tandem triplication of a basic repeating unit. In order to determine the structure of the insert as whole and the functional unit as a transposon, we analyzed nine clones isolated from genomic libraries of W(psm1) and W(cho). The repeating unit of the 16.1 kb insertion was the retrotransposon ninja and the DNA sequence of the entire element was determined. The ninja transposon is 6644 bp in length, with a 316 bp long terminal repeat (LTR) on each end. It contains two openreading frames (ORFs), and the pol region is divided between the two ORFs in contrast the organization of other retrotransposons. An alignment analysis of the reverse transcriptase sequences suggested that the ninja element is the first Drosophila retrotransposon belonging to the Pao subgroup.