Transposable Elements and Genome Size Variations in Plants

Although the number of protein-coding genes is not highly variable between plant taxa, the DNA content in their genomes is highly variable, by as much as 2,056-fold from a 1C amount of 0.0648 pg to 132.5 pg. The mean 1C-value in plants is 2.4 pg, and genome size expansion/contraction is lineage-specific in plant taxonomy. Transposable element fractions in plant genomes are also variable, as low as ~3% in small genomes and as high as ~85% in large genomes, indicating that genome size is a linear function of transposable element content. Of the 2 classes of transposable elements, the dynamics of class 1 long terminal repeat (LTR) retrotransposons is a major contributor to the 1C value differences among plants. The activity of LTR retrotransposons is under the control of epigenetic suppressing mechanisms. Also, genome-purging mechanisms have been adopted to counter-balance the genome size amplification. With a wealth of information on whole-genome sequences in plant genomes, it was revealed that several genome-purging mechanisms have been employed, depending on plant taxa. Two genera, Lilium and Fritillaria, are known to have large genomes in angiosperms. There were twice times of concerted genome size evolutions in the family Liliaceae during the divergence of the current genera in Liliaceae. In addition to the LTR retrotransposons, non-LTR retrotransposons and satellite DNAs contributed to the huge genomes in the two genera by possible failure of genome counter-balancing mechanisms.

Plasticidade muscular no exercício físico / Plasticity in muscular exercise

O objetivo deste estudo foi elaborar uma revisão dos principais mecanismos celulares e moleculares envolvidos no processo de hipertrofia e modulação fenotípica do músculo esquelético, em resposta ao exercício/treinamento físico. Inicialmente, apresentamos uma visão geral do músculo esquelético, com ênfase nas características gerais das fibras musculares e na plasticidade muscular. Em seguida, descrevemos a morfologia e a participação dos mionúcleos e das células satélites durante a hipertrofia muscular, e também, a atuação dos fatores de crescimento sobre a atividade das células satélites. Finalmente, apontamos as principais vias moleculares que mediam as alterações na expressão de proteínas músculo-específicas, de acordo com a especificidade das respostas funcionais ao exercício/treinamento físico.

The aim of this study was prepare a review of the majors cellular and molecular mechanisms involved in hypertrophy and phenotypic modulation of skeletal muscle in response to exercise/physical training. Initially, we present an overview of skeletal muscle, with emphasis on the general characteristics of the muscle fibers and muscle plasticity. Then, we describe the morphology and participation of myonuclei and satellite cells during muscle hypertrophy, and also the action of growth factors on the activity of cells satellites. Finally, we showed the key molecular pathways that mediate changes in the expression of muscle-specific proteins, according to the specificity of functional responses to the exercise/physical training.

Astyanax hastatus Myers, 1928 (Teleostei, Characidae): a new species complex within the genus Astyanax?

Four populations of Astyanax hastatus Myers 1928 from the Guapimirim River basin (Rio de Janeiro State) were analyzed and three distinct cytotypes identified. These cytotypes presented 2n = 50 chromosomes, with 4M+8SM+10ST+28A (Cytotype A), 8M+10SM+14ST+18A (Cytotype B), 6M+8SM+4ST+32A (Cytotype C) and scanty heterochromatin, mainly located throughout pericentromeric regions of several chromosomal pairs. No homologies with the As-51 satellite DNA were observed in the three cytotypes, although all of them presented multiple 18S rDNA sites, as detected by both silver nitrate staining and FISH (fluorescent in situ hybridization). The application of the term "species complex" in Astyanax is discussed from a cytotaxonomic viewpoint.

Hidden heterochromatin: characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.

Genetic variability and efficiency of DNA microsatellite markers for paternity testing in horse breeds from the Brazilian Marajó archipelago

In this study, 15 microsatellite DNA loci used in comparative tests by the International Society for Animal Genetics were applied to the evaluation of genetic diversity and management, and the efficiency of paternity testing in Marajoara horses and Puruca ponies from the Marajó Archipelago. Based on the genotyping of 93 animals, mean allelic diversity was estimated as 9.14 and 7.00 for the Marajoara and Puruca breeds, respectively. While these values are similar to those recorded in most European breeds, mean levels of heterozygosity were much lower (Marajoara 49 percent, Puruca 40 percent), probably as a result of high levels of inbreeding in the Marajó populations. The mean informative polymorphic content of this 15-marker system was over 50 percent in both breeds, and was slightly higher in the Marajoara horses. The discriminative power and exclusion probabilities derived from this system were over 99 percent for both populations, emphasizing the efficacy of these markers for paternity testing and genetic management in the two breeds.

Molecular characterization of SSS139, a new satellite DNA family in sibling species of the Drosophila buzzatii cluster

We characterized sequences of a novel SSS139 RsaI satellite DNA family in Drosophila gouveai and Drosophila seriema, two members of the Drosophila buzzatii cluster (D. repleta group). The sequences were AT-rich (69 percent) with a monomer unit length of about 139 bp and contained two direct subrepeats of 14 bp and 16 bp, suggesting that it might have originated by the duplication of smaller sequences. Southern and dot-blot hybridization analyses also detected SSS139 in other Drosophila buzzatii cluster species (D. koepferae, D. antonietae, D. borborema and D. serido) but not in D. buzzatii. These results agree with the marginal phylogenetic position of D. buzzatii within the D. buzzatii cluster.

Molecular characterization of cotton leaf Curl Multan virus and its satellite DNA that infects Hibiscus rosa-sinensis / 病毒学报

Virus isolate G6 was obtained from Hibiscus rosa-sinensis showing yellow and leaf curl symptoms in Guangzhou, Guangdong Province. The complete nucleotide sequence of DNA-A was determined to be 2 737 nucleotides encoding six potential ORFs. Comparison showed that G6 DNA-A had more than 89% sequence identify with all isolates of Cotton leaf curl Multan virus (CLCuMV) and shared the highest sequence identify (96.1%) with CLCuMV isolate 62. G6 DNA-A had 87.1%-89.8% sequence identity with those of CLCuRV isolates, while less than 87% identities with other begomoviruses. Phylogenetic analysis of G6 DNA-A and selected begomoviruses showed that G6 was most closely related to CLCuMV isolates, and they clustered together as a separate branch. Satellite DNA molecule (G6 DNAbeta) was found to be associated with G6 using the primers beta01 and beta02. G6 DNAbeta contains 1346 nucleotides, with a potential functional ORF (C1) in complementary sense DNA. Pairwise comparison indicated that G6 DNAbeta had the highest sequence identities with CLCuMV DNAbeta (92.1%) and CLCuRV DNAbeta (88.7%), but less than 80% sequence identities with other reported satellite DNA molecules. Phylogenetic analysis indicated that G6 DNAbeta was most closely related to CLCuMV DNAbeta and the two DNAbetas clustered together as a separate branch, and formed the main branch with DNAbeta of CLCuRV and MYVV-Y47. It is concluded that G6 infecting Hibiscus rosa-sinensis is an isolate of CLCuMV.

Chromosomal distribution of the As51 satellite DNA in two species complexes of the genus Astyanax (Pisces, Characidae)

The chromosomal localization of the As51 satellite DNA was identified by fluorescent in situ hybridization (FISH) in specimens of the characid fish Astyanax scabripinnis and Astyanax fasciatus, which are considered species complexes because of their extensive karyotypical and morphological variability. A conserved chromosomal distribution of the As51 satellite, coincident with distal C-banded segments was demonstrated. The alternative interstitial localization of this satellite DNA and possible alterations of its structure suggest that this sequence underwent quantitative, positional and structural variations, as the A. scabripinnis and A. fasciatus complexes diverged.

Evidence of the differentiated structural arrangement of constitutive heterochromatin between two populations of Astyanax scabripinnis (Pisces, Characidae)

The composition of heterochromatin classes along the chromosomes of specimens from two populations of the fish Astyanax scabripinnis was examined using fluorescence banding with GC- and AT-DNA specific fluorochromes and fluorescence in situ hybridization (FISH) with an AT-rich satellite DNA (As51) probe. For the pericentromeric heterochromatin blocks neither GC/AT-DNA specific fluorochromes nor the FISH technique produce any response with chromosomes from either of the populations. On the other hand, the telomeric distal heterochromatin blocks of both populations fluoresced when the FISH technique was applied but showed distinct responses after GC-specific fluorochrome treatments, leading us to propose different structural arrangements of the FISH-positive heterochromatins. Such differences in chromosome banding patterns together with other karyotypic differences suggest differentiation of these populations at taxonomic level.

Identificação de regiões diferencialmente metiladas em câncer de mama através da técnica AP-PCR sensível à metilação (MSAP-PCR) / Identification of differentially methylated regions in breast tumors by methylation sensitive AP-PCR (MSAP-PCR)

A estabilidade do genoma e o perfil normal de expressão gênica são mantidos por um padrão fixo e pré-determinado de metilação do DNA. Esse padrão pode, no entanto, ser alterado nas células tumorais e contribuir na tumorigênese. Neste trabalho, nós utilizamos a técnica de AP-PCR sensível à metilação (MSAP-PCR) com o objetivo de identificar regiões diferencialmente metiladas em tumores de mama. Através desta metodologia, fomos capazes de identificar duas regiões diferencialmente metiladas. O primeiro fragmento de DNA isolado foi mapeado no cromossomo 2q33-34, nas proximidades do gene ADAM23... O tratamento das linhagens celulares MCF-7 e SKBR-3 com o agente desmetilante 5?-aza-2?-deoxycytidina levou a uma reexpressão do gene ADAM23 e a um decréscimo significativo nos níveis de metilação. Uma maior porcentagem de metilação foi verificada em tumores de mama com um estádio mais avançado e a presença de metilação parece estar associada com a presença de linfonodos axilares comprometidos e de metástases, assim como, com a ocorrência de recidivas da doença... Nossos resultados sugerem que a diminuição nos níveis de metilação da região SATR1 é um evento comum em tumores de mama, o qual pode facilitar a ocorrência de rearranjos cromossômicos e contribuir no processo de progressão tumoral. Nossos dados também reforçam a importância da hipometilação global do genoma na tumorigênese e devem ser considerados no desenvolvimento de novos protocolos de tratamento contra o câncer que utilizam agentes desmetilantes...(AU)

Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern. However, this pattern may be altered in tumor cells, contributing to tumorigenesis. In this work, we used the Methylation Sensitive Arbitrarilly Primed - PCR (MSAP-PCR) in arder to identify differentially methylated regions in breast tumors. Using this methodology, we were able to identify two differentially methylated regions. The first isolated DNA fragment was mapped to chromosome 2q33-34, in the proximity of the ADAM23 gene. The members of the ADAM family are cell surface proteins, which present two characteristic domains: the desintegrin domain and the metalloprotease doma in. The ADAM23 protein presents an inactive metalloprotease domain and it is thought to be involved exclusively in cell-cell adhesion. We verified that the promoter region of the ADAM23 gene is hypermethyllated in 66,7°/o of the tumor celllines and in 51,4- 69,2°/o of the analyzed primary breast tumors. The presence of methylation is strongly associated with reductions in both mRNA and protein expression, suggesting that the silencing of ADAM23 gene may be related to alterations in cell adhesion properties and to tumor progression. A threshold of 40-60°/o of methylated dinucleotides within the promoter region is necessary for the complete silencing of the gene. Treatment of MCF-7 and SKBR-3 cell lines with 5'-Aza 2'-deoxycytidine led to a reactivation of ADAM23 gene expression and a marked decrease in the methylation levei. A higher percentage of methylation was observed in breast tumors in advanced stages and the presence of methylation seems to be associated with the presence of positive axillary lymph nades and metastases as well as with disease recurrence. In ali, these results suggest that the methylation of the AM23 gene may be used as a molecular marker in breast tumors.The second DNA fragment isolated was mapped to chromosome 5 and presented a high similarity with a satellite sequence named SA TR 1. Satellite sequences correspond to blocks of tandemly repeated sequences usually located in regions of pericentromeric and/or telomeric heterochromatin. The loss of methylation in satellite sequences is frequently found in tumors and has been associated with an increased frequency of DNA rearrangements and chromosome instability. The loss of methylation in the STAR1 sequence was observed in 63°/o of the breast tumor cell !ines and in 63-87°/o of primary breast tumors. Patients with a ecrease in the leveis of methylation in the SA TR 1 region presented a shorter disease-free survival in relation to patients with normal leveis of methylation (log-rank, p=0.1963). Our results suggest that a decrease in the leveis of methylation in the SATR1 region is common in breast tumors and may facilitate the occurrence of chromosome rearrangements and contribute to tumor progression. Our results also reinforce the importance of global genome hypomethylation in tumorigenesis and must be considered in the development of new treatment protocols, which use demethylating agents in the treatment of cancer (AU)

Hypermethylation of hMLH1 and microsatellite instability in ovarian mucinous tumors / 中国医学科学院学报

<p><b>OBJECTIVES</b>To investigate the role of hMLH1 promoter hypermethylation and microsatellite instability (MSI) in the development of ovarian mucinous tumors.</p><p><b>METHODS</b>One hundred and seven of paraffin-embedded specimens of ovarian mucinous tumors (malignant 49, borderline 35, and benign 23) were collected from Women's Hospital, School of Medicine, Zhejiang University from 1995 to 2001. The assessment of MSI was based on the use of a panel of six microsatellite markers (BAT-25, BAT-26, BAT-40, D5S346, D17S250, and D2S123) by polymerase chain reaction (PCR). Hypermethylation of hMLH1 promoter region was detected using restriction cut analysis.</p><p><b>RESULTS</b>4.3% (1/23), 14.3% (5/35), and 36.7% (18/49) of benign tumors, borderline tumors, and malignant tumors respectively displayed hypermethylation of the hMLH1 promoter. The hMLH1 promoter hypermethylation rate of malignant group was significantly higher than that of borderline and benign group (P = 0.023, 0.004), but no significant difference between the borderline group and the benign group (P = 0.438); 4.3% (1/23), 8.6% (3/35), and 16.3% (8.49) of benign tumors, borderline tumors, and malignant tumors showed MSI positive phenotype. But there were no significant differences each other in the MSI positive phenotype rate; 75% (9/12) MSI positive phenotype ovarian mucinous tumors were hypermethylated at hMLH1 promoter, while the MSI-phenotype tumors were unmethylated in 84.2% (80.95) of cases. There was significant correlation between MSI positive phenotype and hMLH1 promoter hypermethylation (P = 0.000).</p><p><b>CONCLUSIONS</b>In ovarian mucinous tumors, malignant, borderline, and benign tumors exist hMLH1 promoter hypermethylation. Hypermethylation of hMLH1 promoter results MSI in ovarian mucinous tumors. Methylation of hMLH1 promoter and MSI may be involved in the carcinogenesis of ovarian mucinous cancer.</p>

Trypanosoma cruzi: o núcleo em questäo / Trypanosoma cruzi: the nucleus in check

A organizaçäo estrutural do núcleo vem se mostrando cada vez mais fundamental para os processos de metabolismo de DNA e RNA. A vantagem de uma compartimentalizaçäo nuclear é clara, pois permite a concentraçäo de fatores regulatórios estruturais e enzimáticos para estes processos. Se, por um lado, consideravéis evidências sugerem o papel da arquitetura nuclear na coordenaçäo dos processos nucleares, por outro lado, os mecanismos moleculares que estabelecem esta relaçäo ainda näo foram definidos. O T. cruzi é um organismo eucarionte unicelular, com mecanismos de transcriçäo e replicaçäo bastante peculiares e aparentemente menos complexos que aqueles presentes nos demais eucariontes. Nos trypanosomatideos, a transcriçäo é um processo contínuo, produzindo grandes policistrônicas, que säo processadas através de trans-splicing e poliadenilaçäo. Näo há promotores definidos para RNA polimerase II, sendo o controle da expressäo gênica a nível pós-transcricional. Quanto ao processo de replicaçäo, näo foi definida seqüência de origem de replicaçäo. O ciclo de vida do T. cruzi envolve formas replicativas, que näo infectam(amastigotas e epimastigotas) e formas infectivas, que näo replicam(tripomastigotas). Em um primeiro trabalho, nós comparamos a organizaçäo nuclear entre as diferentes formas do parasita e observamos que nas formas replicativas o núcleo é arredondado, contém nucléolo e a heterocromatina está concentrada na periferia nuclear. Por outro lado, nas formas infectivas o núcleo é alongado, o nucléolo näo é evidente e a heterocromatina encontra-se dispersa no espaço nuclear. Esta reorganizaçäo nuclear, que ocorre quando as formas proliferativas transformam-se em infectivas, é acompanhada de uma diminuiçäo geral da atividade transcricional tanto da RNA polimerase I quanto da RNA polimerase II. Apesar de näo haver no T. cruzi uma regulaçäo de transcriçäo aparente, nós mostramos por...

Caracterización de tres microsatelites del gen de fibrosis quistica en familias argentinas / Characterization of 3 microsatellites of the cystic fibrosis gene in Argentine families

La población de Argantina es altamente Heterogénea para las mutaciones del gen regulador de la conductancia de transmembrana de la fibrosis quística (CFTR). El estudio de 14 mutaciones reportadas entre las más frecuentes, detectó ambos alelos mutados (genotipo completo) en sólo el 51 porciento de los pacientes. Este estudio confirmó el diagnóstico de Fibrosis Quística de estos pacientes y posibilitó detectar entres sus familiares, individuos portadores asintomáticos. Sin embargo, en el resto de los pacientes, el análisis molecular directo, no aportó la información necesaria para el asesoriamento familiar. Con el objetivo de estabelecer la transmisióan de microsatélite (IVS17bTA, IVS8CA y IVS17bCA) localizados en regiones intrónicas del gan CFTR. En las 40 familias FQ analizadas, se detectaron diferentes variantes alélicas, 15 para IVS17TA, 10 para IVS8CA y 4 para IVS17CA . El contenido de información polimórfica y de heterocigosis de estos marcadores fue elevado, a exepción de IVS17bCA que resultó poco polimórfico. A través del análisis simultáneo de estos tres microsatélite se pudo asesorar al 100 porciento de las familias. Nuestros resultados demuestran que estos microsatélites, constituyen un excelente gru[po de marcadores, útiles para realizar estudios de ligamiento de población Argentina.

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.

Genetics and colorectal cancer (HNPCC) in adolescence: a case report

Os autores relatam um caso de cancer do colon em uma doente com 17 anos de idade sem polipose ou historia familiar previa. Por tratar-se do primeiro caso desta familia, foi aventada a hipotese de um caso de HNPCC. A reacao da cadeia de polimerase (PCR) do tumor demonstrou alteracoes em quatro regioes polimorficas. A analise de duas destas regioes revelou a perda de material genetico evidenciando-se a presenca de instabilidade, sugerindo HNPCC. Submetida a ileo-retoanastomose e quimioterapia adjuvante com boa evolucao. Os autores discutem a importancia da historia familiar, do estudo genetico, da imuno-histoquimica e da instabilidade de microssatelites em pacientes jovens com cancer colorretal

The Optimization of Human Sperm Decondensation Procedure for Fluorescence in Situ Hybridization / 대한불임학회지

Studies were conducted to determine the efficiency of decondensation protocols. Sperm obtained from seven normal donors was immediately washed after liquefaction and then decondensed using the method of West or al. (1989) and my original protocol. My optimized protocol entailed mixing 1 ml aliquots of semen with 4 ml phosphate buffered saline (PBS) Following centrifugation, pellets were resuspended in 1 ml PBS containing 6 mM EDTA. After centrifugation, pellets were resuspended in 1 ml PBS containing 2 mM dithiothreitol at 37degrees C for 45 min. Following mixing with 2 ml PBS and centrifugation, pellets were resuspended by vortexing. While vortexing, 5 ml of fixative were gently added. Slide preparation was accomplished using the smear method and it was stored at 4degrees C. When comparing these protocols, the degree of sperm decondensation and head swelling was monitored by measuring nuclear length, area, perimeter, and degree of roundness using FISH analysis software. Apparent copy number for chromosome 1 and, separately, for the sex chromosomes was determined by FISH using satellite DNA probes for loci DIZ1, DXZ1 and DYZ3. Sperm treated by my decondensation protocol showed significant increases (p<0.05) in length, area, perimeter, and degree of roundness. There was a significant decrease (p<0.05) in the frequency of nuclei displaying no signal but no change in the frequency of nuclei with two signals in samples decondensed by my protocol.. My data suggested that decondensation using my original protocol may lower the frequency of cells with spurious 'nullisomy' due to hybridization failure without inducing spurious 'disomy' resulting from increased distances between split signals.

A validation study of four single locus probes (MS1, MS31, MS43 and g3) in a Korean population: further evaluation for paternity testing

Hypervariable tandem repetitive regions in human DNA are proving to be increasingly useful for genetic analysis in humans. We chose four single locus probes (SLP; MS1, MS43, MS8 and g3) for a validation test among Koreans. The specimens were from 216 unrelated individuals and 33 paternity inclusion families. Extracted DNA from EDTA blood was restricted by Hinfl and electrophoresed in 0.7% agarose gel, transferred and hybridized with chemiluminescent probes. Heterozygosity was over 90% by all of the probes. Total numbers of unassignable mutant bands from 33 paternity inclusion cases were 5, and the highest mutation rate was determined in probe MS1(0.045). The probability of having the same DNA band between two unrelated individuals was 5.7 x 10(-10) when four SLPs were used at the same time. The data presented here on allele frequencies and mutation rates provide preliminary data supporting the validity of these probes in paternity analysis and forensic investigators in the Korean population.

Construcción de genotecas genómicas de Plasmodium falciparum, selección de clonos que codifiquen para calmodulina y para la secuencis repetitiva PFCOL692, secuenciación de PFCOL692 / Construction of genomic libraries for plasmodium falciparum, selection of clones codifying for calmodulina and the repetitive sequence PFCOL 692, determination of PFCOL 692 sequence

En el presente trabajo se reporta el primer DNA satélite (PFCOL692) aislado para Plasmodium falciparum. La unidad básica es una secuencia de aproximadamente 700 pares de bases (pb). Las dos variantes secuenciadas presentan una homología del 85 por ciento. Los patrones de bandas producidos al hibridar digestiones de DNA del parásito con el DNA satélite marcado radioactivamente, permitieron detectar la existencia de muchas más variantes. Además, las variantes se encuentran a nivel subtelomérico organizadas en bloques de seres unidos cabeza a cola. Por la localización esta secuencia debe jugar un papel importante en los rearreglos que producen polimorfismo cromosómico y antigénico en Plasmodium falciparum. De los patrones de bandas obtenidos por la hibridación de esta secuencia con DNA de diferentes aislados digeridos con enzima de restricción, se pudo ver la utilidad de esta secuencia para caraterizar cepas en el laboratorio. Se construyó una genoteca de Plasmodium falciparum en el fago Lambda EMBL-4 y fueron aislados 28 clonos que contienen la secuencia PFCOL692. El resultado del manejo de restricción de los clonos (en otro trabajo) confirman la organzación planteada. Como otra parte del trabajo se construyó una genoteca de Plasmodium falciparum en el fago Lamda gt10. Se diseño una sonda de oligonucleótidos con base en la secuencia del gen de calmodulina de Plasmodium falciparum. Con la sonda, se aisló un clon que contiene la mayoría del gen de calmodulina del parásito