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Evolutionary conservation and molecular characteristics of repetitive sequences of Drosophila koepferae.

Thirteen middle repetitive DNA clones obtained from the genome of Drosophila koepferae have been tested for their evolutionary conservation in the other seven species of the buzzatii and martensis clusters (repleta group). All but two of these clones exhibit qualitatively similar patterns of hybridization in the eight species. The average interspecific hybridization signal is 85 per cent of that found intraspecifically, ranging from 73 to 93 per cent. Partial sequencing of six of these clones has shown sequences related to the retrotransposon Gypsy, first characterized in D. melanogaster, as well as to the Anopheles gambiae LINE elements T1Ag and Q. A fragment of a hitherto unknown, short inverted repeat transposable element has also been found. The evolutionary conservation of repetitive D. koepferae sequences seems to be related to the high proportion of simple DNA and inactive mobile elements in the genome of this species.

Amino Acid Sequence↗

Formation of novel hairpin structures by telomeric C-strand oligonucleotides.

Telomeres are specialized structures at the ends of chromosomes that are required for long term chromosome stability and replication of the chromosomal terminus. Telomeric DNA consists of simple repetitive sequences with one strand G-rich relative to the other, C-rich, strand. Evolutionary conservation of this feature of telomeric repeat sequences suggests that they have specific structural characteristics involved in telomere function. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoretic analyses showed that telomeric C-strand oligonucleotides form stable non-Watson-Crick hairpin structures containing C.C+ base pairs. Formation of such hairpins may facilitate previously reported G-strand exclusive interactions.

Animals↗

Home cage presentation of complex discrimination tasks to marmosets and rhesus monkeys.

The study reported here demonstrates the feasibility of presenting cognitive tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) to either marmosets or rhesus monkeys in their home cages. This location of testing offers opportunities for the measurement of additional indices, for example spontaneous behaviour (Prowse et al. 1995) and electrophysiology (Pearce et al. 1998) as well as facilitating repeated test presentation. Results from 12 marmosets and 4 rhesus monkeys which have completed several sequences of an eight-stage discrimination task involving simple discriminations, compound discriminations and reversals are reported. The paradigm developed has application in long-term studies. Tests from CANTAB have been used extensively in normal humans (Robbins et al. 1994) as well as a range of patient groups (Owen et al. 1992, Elliott et al. 1995) and to assess drug effects (Coull et al. 1996). Additionally some of these tests have been presented to marmosets (Roberts et al. 1988) to examine neuropsychological functioning. This comparative approach facilitates meaningful cross species comparison, particularly in the study of the effects of pharmacological intervention.

Animals↗

A simple and rapid analysis of triplet repeat diseases by expand long PCR.

It is now known that 15 monogenic, mostly neurological, disorders are caused by the same type of mutations that occur in trinucleotide repeat sequences in certain genes. Since they share a nonspecific and variable phenotype, the accurate diagnosis could be made only by DNA analysis. We developed an Expand Long PCR assay that provides more reliable molecular diagnosis of such disorders. Its main characteristics are robust amplification of expanded alleles, simplicity, low cost and speed. We suggest the use of Expand Long PCR for routine molecular diagnosis of triplet repeat diseases, and present such analysis of the fragile X syndrome, myotonic dystrophy and Huntington's disease.

Base Sequence↗

Construction of silk fiber core in lepidoptera.

The formation and properties of lepidopteran silk fibers depend on amino acid repeats in the principal protein, heavy chain fibroin (H-fibroin). In H-fibroins of the "bombycoid" type, concatenations of alanine or of the GAGAGS crystalline motifs (1st tier repeats) and adjacent sequences breaking periodicity make 2nd tier repeats. Two to six such repeats comprise a 3rd tier assembly, and 12 assemblies, linked by an amorphous sequence, constitute the repetitive H-fibroin region. Heterogeneity in the repeat length and intercalation of amorphous regions prevent excessive crystallization. In the "pyraloid" H-fibroins, iterations of simple motifs are absent and assemblies of several complex motifs constitute highly regular repeats that are organized in about 12 highest order reiterations without specific spacers. Repeat homogeneity appears crucial for the alignment and interaction of the disjunct motifs that must be registered precisely to form crystallites; repeat heterogeneity is associated with decreased fiber strength. Both H-fibroin types are typically hydrophobic, and their secretion requires disulfide linkage to light chain fibroin and participation of another protein, P25. These auxiliary proteins are absent in saturniid moths with amphiphilic H-fibroin repeats. The selection at nucleic acid and protein levels and the availability of nutrients play roles in H-fibroin evolution.

Amino Acid Sequence↗

mm2-ivh: simple and precise overlap detection in alpha satellite HORs with interval hashing.

SUMMARY: We propose a new algorithm, "interval hashing," which distinguishes identical k-mers arising from different repeat sequences, particularly in complex repeat arrays such as alpha satellite HORs. We implement this algorithm as a fork of minimap2, named mm2-ivh. In local assembly of alpha satellite HORs, mm2-ivh accurately reconstructs more haplotypes than assemblers using standard minimizers. AVAILABILITY AND IMPLEMENTATION: mm2-ivh is available under the MIT license at https://github.com/ocxtal/mm2-ivh and runs on common Unix-compatible systems.

Algorithms↗

Human paralogs of KIAA0187 were created through independent pericentromeric-directed and chromosome-specific duplication mechanisms.

KIAA0187 is a gene of unknown function that maps to 10q11 and has been subject to recent duplication events. Here we analyze 18 human paralogs of this gene and show that paralogs of exons 14-23 were formed through satellite-associated pericentromeric-directed duplication, whereas paralogs of exons 1-9 were created via chromosome-specific satellite-independent duplications. In silico, Northern, and RT-PCR analyses indicate that nine paralogs are transcribed, including four in which KIAA0187 exons are spliced onto novel sequences. Despite this, no new genes appear to have been created by these events. The chromosome 10 paralogs map to 10q11, 10q22, 10q23.1, and 10q23.3, forming part of a complex family of chromosome-specific repeats that includes GLUD1, Cathepsin L, and KIAA1099 pseudogenes. Phylogenetic analyses and comparative FISH indicates that the 10q23.1 and 10q23.3 repeats were created in 10q11 and relocated by a paracentric inversion 13 to 27 Myr ago. Furthermore, the most recent duplications, involving the KIAA1099 pseudogenes, have largely been confined to 10q11. These results indicate a simple model for the evolution of this repeat family, involving multiple rounds of centromere-proximal duplication and dispersal through intrachromosomal rearrangement. However, more complex events must be invoked to account for high sequence identity between some paralogs.

Animals↗

[Construction, expression and purification of repeat fragment polymer of oInhibin alpha-subunit N terminal 1-33 fragment].

A cDNA sequence coding for ovine inhibin N terminal 1-33 AA residue fragment (INH) was inserted between BamHI\SacI sites in plasmid pRSET-A to generate plasmid pR-INH. By utilizing a pair of isocaudamer BamHI and Bgl II sites and another downstream Hind III site, following simple double digestions and combination ligation of the resultant products, 2 to 6-repeat INH genes were constructed respectively. Each plamids containing 3 to 6 repeated INH fragment genes, pR-3INH, pR-4INH, pR-5INH and pR-6INH, directed expression of the target proteins in E. coli. BL21 (DE3) under induction of ITPG, which respectively accounted for 6%, 6%, 7% and 8% of the total bacterial protein. The expressed target proteins were all in the form of inclusion bodies. The above results implied that utilization of isocaudamer restriction disgetion sites in expression plasmid is capable of rapidly and correctly constructing repeat fragment polymer of short peptides, which may become a new method in construction of high immunogenic recombinant vaccines of short peptides.

Animals↗

Exceptionally high levels of recombination across the honey bee genome.

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

Animals↗

Organization of the gene encoding the human macrophage mannose receptor (MRC1).

The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.

Amino Acid Sequence↗

Human ribosomal DNA: novel sequence organization in a 4.5-kb region upstream from the promoter.

We have investigated the molecular organization of a portion of the human ribosomal nontranscribed spacer, by determining the sequence of 4580 bp of DNA upstream from the promoter. This region contains two pairs of oppositely oriented Alu elements, each of which is separated by dA (or dT)-rich stretches. One dT-rich region extends over 800 bp and is of variable length in different ribosomal genes. This and other portions of the spacer consist of simple sequences reiterated many times: for instance, (TACAA)26, (TTTC)117, and (TTGC)47. We are able to position the distal junction of the ribosomal repeat and sequences that have the propensity to form alternate structures, such as Z-DNA and bent DNA. A complex DNA methylation pattern and the influence on transcription of analogous regions in other species, suggest that this upstream area may be important to the expression of the human gene.

Base Composition↗

Graftskin therapy in epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a family of 23 genetic skin disorders for which treatments are mainly supportive. Graftskin is a bilayered living human skin construct characterized by a normal expression profile of all the genes reported as mutant in EB. OBJECTIVE: The objective of this study was to evaluate the efficiency and durability of graftskin in the treatment of EB. METHODS: A total of 9 children with EB were treated with graftskin. These include EB simplex: Dowling-Meara type (n = 2); Weber-Cockayne type (n = 1); junctional EB-Herlitz type (n = 1); and recessive dystrophic EB (n = 5). Lesions were debrided of epidermis and crusts followed by application of fenestrated graftskin under sterile conditions. Syndactyly hand release for "mitten deformity" was performed after removal of all epidermis under general anesthesia. All treatment sites were dressed with a nonadherent contact layer followed by absorbent foam dressing, roll gauze, and a compression wrap covering and were left intact for 1 week. Graft take was assessed clinically at weeks 1, 2, 4, 12, and 20 to 28. Graft persistence was assessed by electron microscopy and polymerase chain reaction analysis at weeks 4 and 12, and between weeks 20 and 28 on selected cases. RESULTS: A total of 96 sites were treated with 90% to 100% healing observed by 5 to 7 days, and many sites appearing as normal skin by 10 to 14 days. Finger and hand lesions showed 50% to 90% improvement in range of motion over baseline. Two children learned to walk after graftskin treatment of chronic plantar lesions. Two children had improvement in their chronic anemia after graftskin treatment. All patients and/or parents reported rapid pain resolution. Immunologic and genetic studies of graft persistence revealed evidence of donor DNA up to 28 weeks after graftskin application. None of the samples from female patients demonstrated Y chromosome-specific sequence when analyzed by the method of short tandem repeat. CONCLUSION: The encouraging results reported herein support the hypothesis that graftskin is more than a simple bandage or a source of growth factors to stimulate autologous closure of EB wounds. The improved quality of life and rapid achievement of growth/development milestones we have observed makes this an exciting step forward in the care of the patient with EB.

Adolescent↗

Approaches to half-tetrad analysis in bacteria: recombination between repeated, inverse-order chromosomal sequences.

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.

Bacteria↗

Anticipation of age at onset in panic disorder.

OBJECTIVE: Anticipation (i.e., the decrease in age at onset or the increase in severity of a disorder in successive generations) has recently been reappraised as a key to understanding the genetics of some familial illnesses. The purpose of this study was to search for possible anticipation in panic disorder. METHOD: Thirty-eight unilineal, multigenerational families with multiple directly interviewed members who had panic disorder were compared across two successive generations for 1) age at the first panic attack, 2) age at the onset of panic disorder, and 3) the highest degree of agoraphobia ever experienced, as a tentative index of severity of illness. Intergenerational pairwise comparisons were implemented according to four different sampling schemes: random pairs, random transmitting pairs, all possible pairs, and all possible transmitting pairs. RESULTS: Life table analyses showed a significant decrease in the time before the first episode of panic and onset of panic disorder from the older to the younger generation. Evidence for anticipation was found for both indexes of onset in all four sampling schemes. No evidence of a generational effect on the index of severity of agoraphobia was found. Corrections for possible biases suggested that these results are not likely to be simple artifacts. CONCLUSIONS: Anticipation is supported in this specific set of families and, if it is confirmed by other studies, a role for trinucleotide repeat sequences may be considered to account for the familial aggregation of panic disorder.

Adolescent↗

A model of empathic communication in the medical interview.

OBJECTIVE: To formulate an empirically derived model of empathic communication in medical interviews by describing the specific behaviors and patterns of interaction associated with verbal expressions of emotion. DESIGN: A descriptive, qualitative study of verbal exchanges using 11 transcripts and 12 videotapes of primary care office visits to a total of 21 physicians. SETTING: An urban health maintenence organization (HMO), an urban university-based general medicine clinic, and an urban community hospital general medicine clinic. ANALYTIC METHOD: Individual review of transcripts by each research team member to identify instances of expressed or implied emotional themes and to observe the physicians' responses. Individual ratings were compared in group discussions to achieve consensus about the classifications. Similar consensus-based classification was used for review of videotapes. RESULTS: We observed that patients seldom verbalize their emotions directly and spontaneously, tending to offer clues instead. If invited to elaborate, patients may then express the emotional concern directly, and the physician may respond with an accurate and explicit acknowledgment. In most of the interviews, the physicians allowed both clues and direct expressions of affect to pass without acknowledgment, returning instead to the preceding topic, usually the diagnostic exploration of symptoms. With emotional expression so terminated, some patients attempted to raise the topic again, sometimes repeatedly and with escalating intensity. We noted a parallel dynamic for encounters in which patients sought praise. We summarized the full interactional sequence in a simple descriptive model. CONCLUSIONS: This empirically derived model of empathic communication has practical implications for clinicians and students who want to improve their communication and relationship skills. Based on our observations, the basic empathic skills seem to be recognizing when emotions may be present but not directly expressed, inviting exploration of these unexpressed feelings, and effectively acknowledging these feelings so the patient feels understood. The frequent lack of acknowledgment by physicians of both direct and indirect expressions of affect poses a threat to the patient-physician relationship and warrants further study.

Communication↗

Molecular cloning of the breakpoints of a complex Philadelphia chromosome translocation: identification of a repeated region on chromosome 17.

Complex translocations in chronic myelogenous leukemia involve various chromosomes, in addition to chromosomes 9 and 22, in a nonrandom fashion. We have analyzed the DNA from leukemia cells characterized by a complex translocation, t(9;22;10;17)(q34;q11;p13;q21), by using the techniques of Southern blot hybridization, in situ hybridization, and molecular cloning; one of the breakpoints is at 17q21, a band that is frequently involved in complex 9;22 translocations. All of the breakpoint junctions and the corresponding normal sequences from the four involved chromosomes have been molecularly cloned. Restriction mapping is consistent with a simple concerted exchange of chromosomal material among the four chromosomes, except that additional changes appeared to have occurred within the chromosome 17 sequences. The cloned sequences on chromosome 17 at band q21 were found to be repeated in normal cells. By fluorescence in situ hybridization, a strong signal is seen at 17q21, but a weaker signal is also present at 17q23. By comparison with other primate species, an inversion in chromosome 17 during evolution appears to be responsible for the splitting of the cluster of repeat units in normal human cells.

Animals↗

Repeated sequence elements in the high molecular weight basic nuclear proteins from the winter flounder.

In maturing sperm of the winter flounder, histones are not replaced by protamines but instead joined by a group of high molecular weight basic nuclear proteins. Despite their large size and number of components, these proteins were reduced to a relatively simple set of peptides by a "limit" digestion with endoprotease Lys-C. Nine of these peptides, that together account for half of the mass of the digest, were purified by two rounds of chromatography on a C18 reverse-phase high pressure liquid chromatographic column and analysed by sequential Edman degradation. Their sequences can be divided into two homology groups. Seven of the peptides contain all or part of a dodecapeptide consensus sequence, NH2-Ser-Pro-Met-Arg-Ser-Arg-Ser-Pro-Ser-Arg-Ser-Lys-COOH, which appears to be tandemly repeated. This dodecapeptide contains a previously recognized consensus phosphorylation sequence, NH2-Arg-Ser-Arg-Ser-Pro-COOH, in which both serines are phosphorylated during the early stages of spermiogenesis. The other homology group has the sequence NH2-Arg-Arg-Val-X-X-Pro-Lys-COOH, where X-X is either Gln-Thr or Pro-Ser. The dodecapeptide and heptapeptide sequences form at least 35 and 11%, respectively, of the high molecular weight basic nuclear proteins and are, therefore, repeated many times over in these proteins. A search for identical or homologous sequences within the Protein Sequence Database indicated that they are unique. The closest matches were to protamines and some viral DNA-binding proteins.

Amino Acid Sequence↗

Original domain for the serum albumin family arose from repeated sequences.

The characteristic three-domain structure has been conserved throughout mammalian evolution by serum albumin and its fetal counterpart, alpha-fetoprotein. Thus, one still detects 35.2% amino acid sequence homology between bovine serum albumin and murine alpha-fetoprotein. Yet, natural selection cannot be invoked as the major factor responsible for the observed conservation of these sequences, for the simple reason of their dispensability. Inherited analbuminemia is apparently a harmless trait in man and the rat. The conservation appears inherent in their repetitious origin. Each protein is made of triplicate copies of the ancestral domain. Furthermore, analysis of the published sequence data suggests that the original coding sequence for the ancestral domain arose as repeats of the 18-base-long primordial building block sequence TTC-ACA-GAG-GAG-CAG-CTG specifying Phe-Thr-Glu-Glu-Gln-Leu and its shorter subsidiary TTC-ATG-GAG-GAG specifying Phe-Met-Glu-Glu. Consequently, the homology between bovine serum albumin and alpha-fetoprotein is mostly confined to small segments still specified by recognizable descendants of these building block sequences. The point to be made here is that evolutionary conservation of coding sequences can be an inherent property; natural selection need not be invoked.

Amino Acid Sequence↗