Endogenous retroviral sequences present in the human genome

The common carotid arteries of normal adult rats were investigated electron-microscopically after tannic acid fixation. This fixation technique yields a better demonstrability of the structures of the connective tissue, the basal laminae and the surface coat of the cell membrane. The common carotid artery represents a vessel of the elastic type. The intima consists of an endothelium and a narrow gap of connective tissue (0.1-1 micron) which contains single collagenous fibrils and small elastic structures. This space is only occasionally as wide as 3 microns, especially beneath gaps of the internal elastic membrane. In these areas, single cells and structures of densely packed filaments are additionally observed which can neither be attributed to collagenous fibrils nor to elastic fibres. The intima is demarcated from the outside by an internal elastic membrane (1 micron) which shows a number of gaps. The media exhibits 3 to 4 elastic membranes without gaps. Smooth muscle cells of the contractile type stretch in an oblique direction between these membranes, i.e. they are not arranged in a circular or spiral manner. Most of their process-rich ends are inserted directly into the elastic material and not via a basal lamina. Processes from these smooth muscle cells, collagenous fibrils and elastic fibres are seen in the intercellular spaces. The muscle cells are occasionally interlinked by gap junctions. The basal lamina does not surround the muscle cells continuously. The adventitia contains bundles of collagenous fibrils, fibrocytes, a few small vessels and nerves with a perineuronal envelope. Nerves could not be demonstrated in the media. The oblique course of the smooth muscle cells and the insertion into the elastic membranes indicate that these cells do not predominantly contribute to changes in the width of the lumen but also serve the stabilisation and resetting of the elastic membranes. Contraction is probably induced by an opening of stretch-dependent Ca2+ channels. Due to the interlinkage with gap junctions, the muscle cells of one layer respond as a functional unit. Our findings provide a morphological basis for elucidating commonly encountered changes, such as smooth muscle migration through a normally interrupted inner elastic lamina.     

Duplication and distribution of repetitive elements and non-unique regions in the human genome

The natural history of patients with coronary artery disease and diastolic dysfunction who underwent coronary artery bypass grafting (CABG) is not well known. The aims of our study were to evaluate the incidence of diastolic dysfunction, its evolution after CABG and its possible correlation with adverse in-ICU prognosis. We studied 88 consecutive patients scheduled for CABG with not severely depressed left ventricular function (ejection fraction > 35%) and multivessels disease. Buckberg cardioplegia was used for myocardial protection. Diastolic function was investigated by recording mitral and venous pulmonary flow by transesophageal Doppler echocardiography (TEE). TEE examination was performed in operative room pre and post-bypass, at ICU arrival and after three months. Diastolic dysfunction was defined as mild, moderate and severe. Adverse in ICU events were defined as: use of inotropic drugs or ventricular mechanical support, an ICU stay > 24 hours, perioperative myocardial infarction and death. The study group was compared with a control group. T-Student test was used; a p < 0.05 was considered significant. A reduced diastolic function was present in 77% of patients at baseline examination. Diastolic dysfunction did not worsen significantly after hypothermic cardiac arrest and reperfusion. It persisted during ICU stay and normalized after three months from CABG in the majority of patients (85%). Diastolic failure was not associated with an adverse ICU prognosis (adverse events: 18 versus 13%; p = ns).     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Multiple repetitive DNA sequences in the paracentromeric regions of Arabidopsis thaliana L

An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 x 10(8) LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kg together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.     

Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer's disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent rho degrees (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the rho degrees cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five "AD" missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from rho degrees cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.     

Alphoid repetitive DNA in human chromosomes

The thesis describes the first extensive DNA sequence analysis that demonstrated that the tandemly repeated alphoid DNA in the centromere of the human chromosomes consists of distinct subfamilies and in a number equal to or exceeding the number of chromosomes. The expected presence of only one or a few distinct subfamily on individual chromosomes was supported by the characterization of an extremely well-defined subfamily specific for chromosome 7 and represented in the original collection of subfamilies. The pattern of chromosome-specificity breaks down among the acrocentric chromosomes where chromosomes 13 and 21 were found to share one and chromosomes 14 and 22 to share another specific subfamily. By in situ hybridization these subfamilies were shown not to be shared by other chromosomes. The remarkable pairwise pattern of sequence homogenization was present also in the chimpanzee genome raising the question of its biological role. However, the subfamilies on these human and chimpanzee chromosomes are not orthologous but were shown to originate from two evolutionarily different repeat families. It follows that dramatic sequence evolution has occurred in one or both species during or after separation. The sequence evolution might even occur at a higher rate in humans. This possibility was studied in orthologous alphoid sequences on the X chromosome of humans and the great apes. The analysis supports the general view that our closest relative is the chimpanzee and indicates that the rate of recombination is increased in the human repeat DNA. A "molecular clock" running faster in this DNA may have evolutionary implications. Finally, the usefulness of alphoid subfamilies as chromosome-specific markers is illustrated in a cytogenetic dissection of the centromeric region of Robertsonian translocations. The breakpoints were located to satellite III DNA leaving these chromosomes dicentric. The order of the different tandem DNAs on the p-arm of the acrocentric chromosomes could also be established.     

Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.     

Absence of human herpesvirus 8 and Epstein-Barr virus genome sequences in cutaneous epithelial neoplasms arising in immunosuppressed organ-transplant patients

Recent studies have implicated herpesvirus 8 and Epstein-Barr virus in the development of cutaneous malignancies in immunosuppressed patients. In order to examine the strength of this association, we examined 37 malignant, pre-malignant and benign cutaneous epithelial neoplasms removed from immunosuppressed organ recipients for the presence of human herpesvirus 8 and Epstein-Barr viral genome sequences using polymerase chain reaction (PCR) and in situ hybridization. We examined 2 actinic keratoses, 1 benign keratosis, 11 invasive squamous cell carcinomas, 17 squamous cell carcinomas in situ and 6 basal cell carcinomas. We also examined 4 basal cell carcinomas, 1 invasive squamous cell carcinoma and 3 squamous cell carcinomas in immunocompetent hosts. In contrast to findings reported by other investigators, we were unable to detect viral genome sequences in any of the biopsies examined. Our findings suggest that human herpesvirus 8 and Epstein-Barr virus likely do not play an etiologic role in cutaneous epithelial oncogenesis in immunocompromised patients.     

Retrotransposon-like sequences integrated into the genome of pineapple, Ananas comosus

The efficacy of transcervical recanalization of obstructed postoperative Fallopian tubes was evaluated in 29 patients who were referred for recanalization. Nineteen had strictures at the site of Fallopian tube reconstruction, and five had strictures, three had fistulae, and two had fistulae and strictures at the site of reversal surgery. A 0.014-inch highly flexible guidewire was passed through the obstruction into the ampullary segment, followed by a 1. 1-2.2 Fr bougie catheter to dilate the stricture. After recanalization, the distal tube was studied by selective salpingography. The method was technically successful in 17 of 19 patients with underlying inflammatory disease and resultant postoperative strictures. The tubes remained patent in 12 patients for a period of 12-48 months; three patients conceived, all delivering healthy babies. Significant disease of the distal tubes was present in seven patients. The technique succeeded in three of five patients with postoperative strictures following reversal surgery. One patient subsequently conceived and delivered a healthy baby. The technique failed in all five patients with fistulae complicating reversal surgery. Transcervical recanalization is thus recommended in the management of patients with postoperative strictures with underlying inflammatory obstruction and strictures complicating reversal surgery.     

Tiggers and DNA transposon fossils in the human genome

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.     

Spontaneous and induced minisatellite instability in the human genome

Two patients with recurrent parotid gland carcinoma required subtotal petrosectomy and infratemporal fossa type C approach. To achieve en bloc resection, the ascending mandibular ramus and the entire temporomandibular joint, including the adjacent temporal bone, were removed. An original technique for immediate reconstruction of the infratemporal region, including the glenoid fossa and the ramus of the mandible, is described. Rigid fixation, as well as good functional and aesthetic results, was achieved with autologous calvarial bone and full-thickness rib grafts, allowing the patients to mobilize their jaw very rapidly.     

Internal symmetry and repetitive sequences in the primary structure of IRS-proteins, endogenous substrates of insulin receptor

A new method is developed for identification of mirror type internal symmetry in protein primary structures (named as method of internal symmetry scanning). As distinct from our earlier developed graphic method, the application of the new method allows, to identify enough fast and effective, symmetrical segments in proteins of any length, and to determine the type of internal symmetry centres (one or two amino acid residues). By this method the structure of IRS-proteins, endogenous substrates of tyrosine kinase receptors, was analysed. It has been shown that the density of internal symmetry centre distribution and the homology of antiparallel sequences in functionally important regions, which possess conservation of the primary structure (the conservative profile for IRS1-proteins was calculated in this work), are higher in comparison with the variable regions. Groups of repeating sequences in the primary structure of IRS1-proteins are found. The localization of the repeats coincides with one of symmetrical structures. These findings are in line with Chipens' hypothesis in which he established a correlation between the internal symmetry and the homologous repeats.     

Systematic fold recognition analysis of the sequences encoded by the genome of Mycoplasma pneumoniae

A robust tool for fold recognition was applied to the systematic analysis of the sequences below 200 residues encoded by the genome of Mycoplasma pneumoniae. The goal was to determine the additional information gain achievable in genome analysis by fold recognition, beyond the intrinsic limits of homology studies. A list of 124 sequences encoding for soluble proteins or domains not homologous to each other, or to proteins with known three-dimensional structure, was analyzed, resulting in significant Z scores for the energy of the structural models in 12 of these cases. This result indicates that systematic application of fold recognition techniques to the analysis of structurally unassigned soluble proteins can lead to high-confidence structural predictions with an efficiency of about 10%, a relevant contribution besides the complementary approach of homology analysis. Four of the predictions presented include mapping of the putative active site of the target sequence and lead to the detection of probable catalytic and binding residues. The data are discussed with reference to the functional implications of the structural models and to the results reported for the homologous genome of Mycoplasma genitalium.     

Toward a cDNA map of the human genome

Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosome band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for teh rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to map 41 cDNAs with an average insert size of <2 kb to single human chromosome bands. The result provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular signal-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphatase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (2-5 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases.     

Interspersion of different repeated sequences in the wheat genome revealed by interspecies DNA/DNA hybridisation

The repeated sequences in oats DNA have been used to study chromosomal repeated sequence organisation in wheat. Approximately 75% of the wheat genome consists of repeated sequences but only approximately 20% will form heteroduplexes with repeated sequences from oats DNA at 60 degrees C in 0.18 M Na+. The proportion of wheat DNA that forms heteroduplexes with oats DNA is shown to be independent of the wheat DNA fragment length. However, the proportion of wheat DNA that is retained with the heteroduplexes when fractionated on hydroxyapatite is very dependent upon the wheat fragment length up to 3500 nucleotides. This is because more non-renatured wheat DNA is attached to the heteroduplexes with longer fragments. The results indicate that the repeated sequences in the wheat genome homologous to repeated sequences in oats are not clustered in the chromosomes but distributed amongst other repeated and possible non-repeated sequences.