Epidemiología genómica y paraparesia espástica tropical asociada a la infección por el virus linfotrópico humano de células T tipo 1 / Genome epidemiology and tropical spastic paraparesis associated with human T-cell lymphotropic virus type 1

OBJETIVO: Caracterizar el ambiente genómico de las secuencias adyacentes al virus linfotrópico humano de células T tipo 1 (HTLV-1) en pacientes con paraparesia espßstica tropical y mielopatía asociada a la infección con HTLV-1 (PET/MAH) de diferentes regiones de Colombia y del Japón. MÉTODOS: Se enfrentaron 71 clones recombinantes con secuencias del genoma humano adyacentes al 5'-LTR de pacientes con PET/MAH, a las bases de datos del Genome Browser y del Gen-Bank. Se identificaron y analizaron estadísticamente 16 variables genómicas estructurales y composicionales mediante el programa informßtico R, versión 2.8.1, en una ventana de 0,5 Mb. RESULTADOS: El 43,0 por ciento de los provirus se localizaron en los cromosomas del grupo C; 74 por ciento de las secuencias se ubicaron en regiones teloméricas y subteloméricas (P < 0,05). Un anßlisis de conglomerados permitió establecer las relaciones jerßrquicas entre las características genómicas incluidas en el estudio; el anßlisis de componentes principales identificó las componentes que definieron los ambientes genómicos preferidos para la integración proviral en casos de PET/MAH. CONCLUSIONES: El HTLV-1 se integró con mayor frecuencia en regiones de la cromatina ricas en islas de citocina fosfato guanina (CpG), de alta densidad de genes y de repeticiones tipo LINE (elemento disperso largo [long interspersed element]) y transposones de ADN que, en conjunto, conformarían los ambientes genómicos blanco de integración. Este nuevo escenario promoverß cambios sustanciales en el campo de la salud pública y en el manejo epidemiológico de las enfermedades infecciosas, y permitirß desarrollar potentes herramientas para incrementar la eficiencia de la vigilancia epidemiológica.

OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0 percent of the proviruses were located in the group C chromosomes; 74 percent of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Incongruent evolution of chromosomal size in rice

To investigate genome size evolution, it is usually informative to compare closely related species that vary dramatically in genome size. A whole genome duplication (polyploidy) that occurred in rice (Oryza sativa) about 70 million years ago has been well documented based on current genome sequencing. The presence of three distinct duplicate blocks from the polyploidy, of which one duplicated segment in a block is intact (no sequencing gap) and less than half the length of its syntenic duplicate segment, provided an excellent opportunity for elucidating the causes of their size variation during the post-polyploid time. The results indicated that incongruent patterns (shrunken, balanced and inflated) of chromosomal size evolution occurred in the three duplicate blocks, spanning over 30 Mb among chromosomes 2, 3, 6, 7, and 10, with an average of 20.3% for each. DNA sequences of chromosomes 2 and 3 appeared to had become as short as about half of their initial sequence lengths, chromosomes 6 and 7 had remained basically balanced, and chromosome 10 had become dramatically enlarged (approximately 70%). The size difference between duplicate segments of rice was mainly caused by variations in non-repetitive DNA loss. Amplification of long terminal repeat retrotransposons also played an important role. Moreover, a relationship seems to exist between the chromosomal size differences and the nonhomologous combination in corresponding regions in the rice genome. These findings help shed light on the evolutionary mechanism of genomic sequence variation after polyploidy and genome size evolution.

Periodic Explosive Expansion of Human Retroelements Associated with the Evolution of the Hominoid Primate

Five retroelement families, L1 and L2 (long interspersed nuclear element, LINE), Alu and MIR (short interspersed nuclear element, SINE), and LTR (long terminal repeat), comprise almost half of the human genome. This genome-wide analysis on the time-scaled expansion of retroelements sheds light on the chronologically synchronous amplification peaks of each retroelement family in variable heights across human chromosomes. Especially, L1s and LTRs in the highest density on sex chromosomes Xq and Y, respectively, disclose peak activities that are obscured in autosomes. The periods of young L1, Alu, LTR, and old L1 peak activities calibrated based on sequence divergence coincide with the divergence of the three major hominoid divergence as well as early eutherian radiation while the amplification peaks of old MIR and L2 account for the marsupial-placental split. Overall, the peaks of autonomous LINE (young and old L1s and L2s) peaks and non-autonomous SINE (Alus and MIRs) have alternated repeatedly for 150 million years. In addition, a single burst of LTR parallels the Cretaceous-Tertiary (K-T) boundary, an exceptional global event. These findings suggest that the periodic explosive expansions of LINEs and SINEs and an exceptional burst of LTR comprise the genome dynamics underlying the macroevolution of the hominoid primate lineage.

Clinical profile and frequency of delta f508 mutation in Indian children with cystic fibrosis.

OBJECTIVE: To document clinical profile of cystic fibrosis (CF) in Indian children and the prevalence of delta F508 mutation in these patients. DESIGN: Observational study. Setting:Pediatric chest clinic in an urban tertiary care center in north India. PERIOD OF STUDY: July 1995 to June 2002. METHODS: Clinical features of 120 children diagnosed as CF by quantitative pilocarpine iontophoresis were recorded. A polymerase chain reaction based test for identification of delta F 508 mutation was performed on all children. RESULTS: Out of 3500 new cases registered in Pediatric Chest Clinic during this period 120, (3.5%) children were diagnosed as CF. Origin of parents of patients traced from almost all the States of north India. Family history suggestive of CF was present in 41 (34%) and consanguinity in 19 (61%) patients. Common clinical manifestations at the time of presentation included recurrent or persistent pneumonia in 118 (98%), failure to thrive in 108 (90%), malabsorption in 96 (80%), history of meconium ileus in 10 (8%), and rectal prolapse was present in 16 (13%). History of salt craving, salty taste on kissing and skin rashes was present in 5 patients each. 49(41%) patients were severely malnourished. Nasal polyposis was present in 5 (4%) patients. Examination of chest revealed evidence of hyperinflation in 100 (83%), kyphosis 20 (17%), crepitations 110 (92%), wheezing 40 (25%) and bronchial breathing in 20 (17%) patients. Average clinical CF scores were 51 (95%; CI 20-80). 48 (40%) patients had a CF score of LT40. Pseudomonas spp was cultured from respiratory secretions of 51 (42%), Staphylococcus spp in 18 (15%), Klebsiella spp in 8 (7%) and Hemophilus influenzae in 2 (2%) patients. Delta F508 mutation was positive in 45 chromosomes out of 240 tested. Patients originated from Pakistan had more frequency of delta F508 mutations. CONCLUSIONS: Cystic fibrosis does occur in Indian children; clinical features are classical. Diagnosis is often delayed and the disease is advanced in most patients at the time of diagnosis. Frequency of Delta F508 mutation is 19% i.e., less than that seen in Caucasian population. There is need to create awareness about occurrence of CF in Indian children.

Evolutionary course of CsRn1 long-terminal-repeat retrotransposon and its heterogeneous integrations into the genome of the liver fluke, Clonorchis sinensis

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their LTR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.