Göran Sperber

Automated recognition of retroviral sequences in genomic data—RetroTector©

Eukaryotic genomes contain many endogenous retroviral sequences (ERVs). ERVs are often severely mutated, therefore difficult to detect. A platform independent (Java) program package, RetroTector© (ReTe), was constructed. It has three basic modules: (i) detection of candidate long terminal repeats (LTRs), (ii) detection of chains of conserved retroviral motifs fulfilling distance constraints and (iii) attempted reconstruction of original retroviral protein sequences, combining alignment, codon statistics and properties of protein ends. Other features are prediction of additional open reading frames, automated database collection, graphical presentation and automatic classification. ReTe favors elements >1000-bp long due to its dependence on order of and distances between retroviral fragments. It detects single or low-copy-number elements. ReTe assigned a ‘retroviral’ score of 890–2827 to 10 exogenous retroviruses from seven genera, and accurately predicted their genes. In a simulated model, ReTe was robust against mutational decay. The human genome was analyzed in 1–2 days on a LINUX cluster. Retroviral sequences were detected in divergent vertebrate genomes. Most ReTe detected chains were coincident with Repeatmasker output and the HERVd database. ReTe did not report most of the evolutionary old HERV-L related and MalR sequences, and is not yet tailored for single LTR detection. Nevertheless, ReTe rationally detects and annotates many retroviral sequences.

Evolution of human endogenous retroviral sequences: a conceptual account.

Abstract.Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic ‘junk’.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a Multi-author Review)

Classification and nomenclature of endogenous retroviral sequences (ERVs): Problems and recommendations

The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.

Cardiac Gated MR Imaging of Cerebrospinal Fluid Flow

This is a preliminary investigation of the cerebrospinal fluid (CSF) spaces using cardiac gated magnetic resonance imaging. A variation of intensity of the signal from the cerebral aqueduct is demonstrated during the cardiac cycle. The pattern of this variation suggests pulsatile CSF flow. Calculations that have been verified by phantom measurements show that CSF flow rates less than I mm/s may be detectable. Magnetic resonance may therefore offer a new method for the demonstration and measurement of CSF flow.

A method for MR quantification of flow velocities in blood and CSF using interleaved gradient-echo pulse sequences

The aim of this study was to establish a rapid method for in vivo quantification of a large range of flow velocities using phase information. A basic gradient-echo sequence was constructed, in which flow was encoded along the slice selection direction by variation of the amplitude of a bipolar gradient without changes in sequence timings. The influence of field inhomogeneities and eddy currents was studied in a 1.5 T interleaved sequences for calibration and in vivo flow determination were constructed, and flow information was obtained by pairwise subtraction of velocity-encoded from velocity non-encoded phase images. Calibration was performed in a nongated mode using flow phantoms, and the results were compared with theoretically calculated encoding efficiencies. In vivo flow was studied in healthy volunteers in three different areas using cardiac gating; central blood flow in the great thoracic vessels, peripheral blood flow in the popliteal vessels, and flow of cerebrospinal fluid (CSF) in the cerebral aqueduct. The results show good agreement with results obtained with other techniques. The proposed method for flow determination was shown to be rapid and flexible, and we thus conclude that it seems well suited for routine clinical MR examinations.

Blood flow and glucose consumption in the optic nerve, retina and brain: Effects of high intraocular pressure

Glucose consumption and regional blood flow were determined using the [14C]-2-deoxyglucose (2-DG) method and microspheres in the optic nerve, the retina and different parts of the brain in monkeys. The relationship between the 2-DG accumulation and blood flow in the optic nerve head region was similar to that in grey matter of the brain under pentobarbital anaesthesia as well as under urethan anaesthesia. Pentobarbital anaesthesia resulted in lower values for blood flow and glucose metabolism in most regions. In the optic nerve the highest values were observed in the distal part; there was a fall in blood flow and metabolism along the nerve. There was a corresponding increase in myelin content. Artificial increments in intraocular pressure resulting in a perfusion pressure (mean arterial pressure minus intraocular pressure) of 40 cm H2O had no appreciable effect on the 2-DG accumulation. At a perfusion pressure of 20 cm H2O 2-DG accumulation in the retina and prelaminar part of the optic nerve was markedly increased indicating partial ischemia resulting in anaerobic glycolysis. At intraocular pressures higher than the systolic arterial blood pressure there was still some accumulation of 2-DG in the intraocular tissues, but no blood flow, which indicates that glucose could diffuse into the eye through the sclera. Behind the lamina cribrosa there was no indication of a reduction in blood flow or a metabolic disturbance. The results indicate that the blood flow and metabolism of the retina and prelaminar part of the optic nerve is disturbed only at very high intraocular pressures, and that even at extreme pressures there is no disturbance behind the lamina cribrosa in acute experiments. The 2-DG method will be useful in further studies on the nutritional status of the optic nerve head since it can detect abnormal glycolysis even in very discrete regions due to its high spatial resolution.

Magnetic Resonance Imaging in Primary Amyloidosis

Twelve patients with primary amyloidosis (AL) were investigated with magnetic resonance imaging (MRI). In 9 patients an abnormal thickening of the heart walls was present and in 2 macroglossia was found at MRI. T1 was significantly increased in liver (p<0.05) and subcutaneous fat (p<0.01) while it was decreased in the spleen (p<0.05). T2 was significantly decreased (p<0.01) in the spleen in patients with amyloidosis, while it was not significantly altered in the liver or subcutaneous fat. After therapy T1 of the liver was reduced towards normal values in 4 patients. It is concluded that MRI might be a method to quantitate the amount of amyloid deposits in the tissue, and that the effect of therapy may be monitored with this technique.

A method for near-continuous determination of aqueous humor flow; Effects of anaesthetics, temperature and indomethacin

A method is described for near-continuous determination of aqueous humor flow. The anterior chamber is perfused with push-pull coupled syringes at a low rate with a fluid containing labelled albumin. An external circuit is used to determine continuously the anterior chamber concentration of the labelled protein. The dilution data are analysed on-line by a minicomputer which permits rapid calculation of the anterior chamber volume and the rate of flow of aqueous humor. The technique and some experiments of technical interest are reported. Experiments in monkeys with different anaesthetics resulted in flow values of 0.99 +/- 0.02, 1.47 +/- 0.09 and 0.99 +/- 0.04 microliter min-1 for pentobarbital, urethane and ketamine anaesthesia, respectively. By using 125I-labelled albumin in one eye and 131I-labelled albumin in the other, it was possible to determine flow in both eyes. Highly significant correlation coefficients between the two sides were found for the rate of aqueous flow, intraocular pressure and anterior chamber volume. Rapid changes in inflow into the anterior chamber from the posterior chamber were produced by elevating and then lowering the intraocular pressure; the delay inherent in the method was about 6 min. Indomethacin, 3 mg kg-1 body wt., had no effect on aqueous humor flow in eyes cannulated with a minimum of trauma. In eyes with problematic cannulation indomethacin at this dose tended to delay an irritation response. Changes in temperature of the fluid perfused through the anterior chamber had no clear effect on the rate of aqueous flow. Warming the animals about 3-4 degrees C above the normal temperature tended to increase the rate of aqueous flow. Cooling by 3-4 degrees C had no clear effect. Cooling after an initial warming also had no clear effect. The rate of flow of aqueous humor from the anterior chamber to the general circulation was calculated from data for the accumulation of labelled albumin in the general circulation. The difference between the rate of aqueous flow determined from the dilution data and the flow into blood was assumed to represent uveoscleral flow. In 14 animals with an aqueous flow of 1.19 +/- 0.08 microliters min-1 the flow to the general circulation was 0.57 +/- 0.055 and uveoscleral flow 0.61 +/- 0.09 microliters min-1. The procedure and mathematical treatment will be applicable to flow determinations with other large molecules and in other systems.

ERV3 and Related Sequences in Humans: Structure and RNA Expression

ABSTRACT The ERV3 locus at chromosome 7q11 is a much studied human endogenous retroviral (HERV) sequence, owing to an env open reading frame (ORF) and placental RNA and protein expression. An analysis of the human genome demonstrated that ERV3 is one of a group of 41 highly related elements (ERV3-like HERVs) which use proline, isoleucine, or arginine tRNA in their primer binding sites. In addition to elements closely related to ERV3, the group included the previously known retinoic acid-inducible element, RRHERVI, also referred to as HERV15, but was separate from the related HERV-E elements. The ERV3-like elements are defective. The only element with an ORF among gag, pro, pol, and env genes was the env ORF of the original ERV3 locus. A search in dbEST revealed ERV3 RNA expression in placenta, skin, carcinoid tumor, and adrenal glands. Expression was also studied with newly developed real-time quantitative PCRs (QPCR) of ERV3 and HERV-E(4-1) env sequences. Results from a novel histone 3.3 RNA QPCR result served as the expression control. QPCR results for ERV3 were compatible with previously published results, with a stronger expression in adrenal gland and placenta than in 15 other human tissues. The expression of the envelope (env) of ERV3 at chromosome 7q11 was also studied by using stringent in situ hybridization. Expression was found in corpus luteum, testis, adrenal gland, Hassals bodies in thymus, brown fat, pituitary gland, and epithelium of the lung. We conclude that ERV3 env is most strongly expressed in adrenal and sebaceous glands as well as in placenta.

Sequence Variability, Gene Structure, and Expression of Full-Length Human Endogenous Retrovirus H

ABSTRACT Recently, we identified and classified 926 human endogenous retrovirus H (HERV-H)-like proviruses in the human genome. In this paper, we used the information to, in silico, reconstruct a putative ancestral HERV-H. A calculated consensus sequence was nearly open in all genes. A few manual adjustments resulted in a putative 9-kb HERV-H provirus with open reading frames (ORFs) in gag, pro, pol, and env. Long terminal repeats (LTRs) differed by 1.1%, indicating proximity to an integration event. The gag ORF was extended upstream of the normal myristylation start site. There was a long leader (including a “pre-gag” ORF) region positioned like the N terminus of murine leukemia virus (MLV) “glyco-Gag,” potentially encoding a proline- and serine-rich domain remotely similar to MLV pp12. Another ORF, starting inside the 5′ LTR, had no obvious similarity to known protein domains. Unlike other hitherto described gammaretroviruses, the reconstructed Gag had two zinc finger motifs. Alternative splicing of sequences related to the HERV-H consensus was confirmed using dbEST data. env transcripts were most prevalent in colon tumors, but also in normal testis. We found no evidence for full length env transcripts in the dbEST. HERV-H had a markedly skewed nucleotide composition, disfavoring guanine and favoring cytidine. We conclude that the HERV-H consensus shared a gene arrangement common to gammaretroviruses with gag separated by stop codon from pro-pol in the same reading frame, while env resides in another reading frame. There was also alternative splicing. HERV-H consensus yielded new insights in gammaretroviral evolution and will be useful as a model in studies on expression and function.