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Fulltext Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA

Murulitharan K, Yusoff K, Omar AR, Peeters BPH, Molouki A

Curr Microbiol, 2021 Apr;78(4):1458-1465.
PMID: 33660046 DOI: 10.1007/s00284-021-02421-z

Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.

Matched MeSH terms: DNA, Complementary/genetics
Cloning and localization of immediate early response 2 (ier2) gene in the brain of medaka

Moriya S, Chourasia D, Ng KW, Khel NB, Parhar IS

J. Chem. Neuroanat., 2016 11;77:24-29.
PMID: 27134039 DOI: 10.1016/j.jchemneu.2016.04.005

Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.

Matched MeSH terms: DNA, Complementary/genetics
Isolation and characterization of oil palm constitutive promoter derived from ubiquitin extension protein (uep1) gene

Masura SS, Parveez GK, Ismail I

N Biotechnol, 2010 Sep 30;27(4):289-99.
PMID: 20123048 DOI: 10.1016/j.nbt.2010.01.337

The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm. We have isolated and characterized the 5' region of the oil palm uep1 gene, which contains an 828 bp sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector, which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional analysis of the promoter through transient expression studies. It was found that the 5' region of uep1 functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested, including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf, mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was demonstrated to be capable of driving gusA gene expression in tobacco.

Matched MeSH terms: DNA, Complementary/genetics
Sequence and localization of kcnk10a in the brain of adult zebrafish (Danio rerio)

Loganathan K, Moriya S, Sivalingam M, Ng KW, Parhar IS

J. Chem. Neuroanat., 2017 Dec;86:92-99.
PMID: 29074372 DOI: 10.1016/j.jchemneu.2017.10.004

kcnk10a has been predicted in zebrafish to be a member of the two-pore domain potassium ion (K+) channel-related K+ (TREK) channel family known as a thermoreceptor. Since reproduction is affected by temperature, Kcnk10a could be involved in the regulation of reproduction. However, expression of kcnk10a in the zebrafish brain and association with reproduction has not been identified. In this study, the full length sequence and localization of kcnk10a in the brain was investigated and gene expressions of the TREK channel family were examined to investigate association with reproduction. We initially identified the full length cDNA sequence of kcnk10a using Rapid Amplification of cDNA Ends and localization in the zebrafish brain using in situ hybridization. Furthermore, we examined the gene expression differences of kcnk2b, kcnk10a and kcnk10b mRNA between genders as well as developmental stages by real-time PCR. The deduced amino acid sequence of the identified kcnk10a mRNA contains highly conserved two pore domains and four transmembrane regions and was higher similarity to zebrafish Kcnk10b than zebrafish Kcnk2a and 2b. kcnk10a mRNA was widely distributed in the brain such as the preoptic area, hypothalamus and the midbrain. kcnk10a mRNA expression exhibited significant difference between mature male and female, and increase during puberty. Kcnk10a could be involved in the regulation of reproductive function.

Matched MeSH terms: DNA, Complementary/genetics
Induced expression of Ganoderma boninense Lanosterol 14α-Demethylase (ERG11) during interaction with oil palm

Lim FH, Rasid OA, Idris AS, As'wad AWM, Vadamalai G, Parveez GKA, et al.

Mol Biol Rep, 2023 Mar;50(3):2367-2379.
PMID: 36580194 DOI: 10.1007/s11033-022-08131-4

BACKGROUND: The basidiomycete fungus, Ganoderma boninense is the main contributor to oil palm Basal Stem Rot (BSR) in Malaysia and Indonesia. Lanosterol 14α-Demethylase (ERG11) is a key enzyme involved in biosynthesis of ergosterol, which is an important component in the fungal cell membrane. The Azole group fungicides are effective against pathogenic fungi including G. boninense by inhibiting the ERG11 activity. However, the work on molecular characterization of G. boninense ERG11 is still unavailable today.
METHODS AND RESULTS: This study aimed to isolate and characterize the full-length cDNA encoding ERG11 from G. boninense. The G. boninense ERG11 gene expression during interaction with oil palm was also studied. A full-length 1860 bp cDNA encoding ERG11 was successfully isolated from G. boninense. The G. boninense ERG11 shared 91% similarity to ERG11 from other basidiomycete fungi. The protein structure homology modeling of GbERG11 was analyzed using the SWISS-MODEL workspace. Southern blot and genome data analyses showed that there is only a single copy of ERG11 gene in the G. boninense genome. Based on the in-vitro inoculation study, the ERG11 gene expression in G. boninense has shown almost 2-fold upregulation with the presence of oil palm.
CONCLUSION: This study provided molecular information and characterization study on the G. boninense ERG11 and this knowledge could be used to design effective control measures to tackle the BSR disease of oil palm.

Matched MeSH terms: DNA, Complementary/genetics
Interethnic variability of warfarin maintenance requirement is explained by VKORC1 genotype in an Asian population

Lee SC, Ng SS, Oldenburg J, Chong PY, Rost S, Guo JY, et al.

Clin. Pharmacol. Ther., 2006 Mar;79(3):197-205.
PMID: 16513444

Chinese and Malay subjects have been reported to require less maintenance warfarin than Indians that could not be accounted for by cytochrome P450 (CYP) 2C9 variants. Vitamin K epoxide reductase complex 1 (VKORC1) is the target enzyme of warfarin, and VKORC1 intronic variants and haplotypes have recently been shown to influence VKORC1 activity and warfarin requirements.

Matched MeSH terms: DNA, Complementary/genetics
Molecular characterization of serotype A foot-and-mouth disease viruses circulating in Vietnam in 2009

Le VP, Nguyen T, Lee KN, Ko YJ, Lee HS, Nguyen VC, et al.

Vet Microbiol, 2010 Jul 29;144(1-2):58-66.
PMID: 20097490 DOI: 10.1016/j.vetmic.2009.12.033

Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in Vietnam in recent years. In this work, six serotype A foot-and-mouth disease viruses (FMDV), collected from endemic outbreaks during January and February of 2009 in four different provinces in Vietnam, were genetically characterized for their complete genome sequences. Genetic analysis based on the complete viral genome sequence indicated that they were closely related to each other and shared 99.0-99.8% amino acid (aa) identity. Genetic and deduced aa analysis of the capsid coding gene VP1 showed that the six Vietnamese strains were all classified into the genotype IX from a total of 10 major genotypes worldwide, sharing 98.1-100% aa identity each other. They were most closely related to the type A strains recently isolated in Laos (A/LAO/36/2003, A/LAO/1/2006, A/LAO/6/2006, A/LAO/7/2006, and A/LAO/8/2006), Thailand (A/TAI/2/1997 and A/TAI/118/1987), and Malaysia (A/MAY/2/2002), sharing 88.3-95.5% nucleotide (nt) identities. In contrast, Vietnamese type A strains showed low nt identities with the two old type A FMDVs, isolated in 1960 in Thailand (a15thailand iso43) and in 1975 in the Philippines (aphilippines iso50), ranging from 77.3 to 80.9% nt identity. A multiple alignment based on the deduced amino acid sequences of the capsid VP1 coding gene of type A FMDV revealed three amino acid substitutions between Vietnamese strains and the strains of other Southeast Asian countries (Laos, Thailand, Malaysia, and the Philippines). Alanine was replaced by valine at residue 24, asparagine by arginine at residue 85, and serine by threonine at residue 196. Furthermore, type A FMDV strains recently isolated in Vietnam, Laos, Thailand, and Malaysia all have one amino acid deletion at residue 140 of the capsid VP1 protein compared with the two old type A FMDV strains from Thailand and the Philippines as well as most other type A representatives worldwide. This article is the first to report on the comprehensive genetic characterization of type A FMDV circulating in Vietnam.

Matched MeSH terms: DNA, Complementary/genetics
Fulltext Construction of an infectious cDNA clone of Enterovirus 71: insights into the factors ensuring experimental success

Lazouskaya NV, Palombo EA, Poh CL, Barton PA

J Virol Methods, 2014 Mar;197:67-76.
PMID: 24361875 DOI: 10.1016/j.jviromet.2013.12.005

Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.

Matched MeSH terms: DNA, Complementary/genetics*
Granulometric analysis of spots in DNA microarray images

Latha B, Venkatesh B

Genomics Proteomics Bioinformatics, 2004 Nov;2(4):222-36.
PMID: 15901251

As the topological properties of each spot in DNA microarray images may vary from one another, we employed granulometries to understand the shape-size content contributed due to a significant intensity value within a spot. Analysis was performed on the microarray image that consisted of 240 spots by using concepts from mathematical morphology. In order to find out indices for each spot and to further classify them, we adopted morphological multiscale openings, which provided microarrays at multiple scales. Successive opened microarrays were subtracted to identify the protrusions that were smaller than the size of structuring element. Spot-wise details, in terms of probability of these observed protrusions, were computed by placing a regularly spaced grid on microarray such that each spot was centered in each grid. Based on the probability of size distribution functions of these protrusions isolated at each level, we estimated the mean size and texture index for each spot. With these characteristics, we classified the spots in a microarray image into bright and dull categories through pattern spectrum and shape-size complexity measures. These segregated spots can be compared with those of hybridization levels.

Matched MeSH terms: DNA, Complementary/genetics
A review of Nipah and Hendra viruses with an historical aside

Ksiazek TG, Rota PA, Rollin PE

Virus Res, 2011 Dec;162(1-2):173-83.
PMID: 21963678 DOI: 10.1016/j.virusres.2011.09.026

The emergence of Hendra and Nipah viruses in the 1990s has been followed by the further emergence of these viruses in the tropical Old World. The history and current knowledge of the disease, the viruses and their epidemiology is reviewed in this article. A historical aside summarizes the role that Dr. Brian W.J. Mahy played at critical junctures in the early stories of these viruses.

Matched MeSH terms: DNA, Complementary/genetics
Prolactin receptor mRNA is upregulated in discus fish (Symphysodon aequifasciata) skin during parental phase

Khong HK, Kuah MK, Jaya-Ram A, Shu-Chien AC

Comp Biochem Physiol B Biochem Mol Biol, 2009 May;153(1):18-28.
PMID: 19272315 DOI: 10.1016/j.cbpb.2009.01.005

Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.

Matched MeSH terms: DNA, Complementary/genetics
RNomic identification and evaluation of npcTB_6715, a non-protein-coding RNA gene as a potential biomarker for the detection of Mycobacterium tuberculosis

Kanniappan P, Ahmed SA, Rajasekaram G, Marimuthu C, Ch'ng ES, Lee LP, et al.

J Cell Mol Med, 2017 10;21(10):2276-2283.
PMID: 28756649 DOI: 10.1111/jcmm.13148

Technological advances in RNA biology greatly improved transcriptome profiling during the last two decades. Besides the discovery of many small RNAs (sRNA) that are involved in the physiological and pathophysiological regulation of various cellular circuits, it becomes evident that the corresponding RNA genes might also serve as potential biomarkers to monitor the progression of disease and treatment. sRNA gene candidate npcTB_6715 was previously identified via experimental RNomic (unpublished data), and we report its application as potential biomarker for the detection of Mycobacterium tuberculosis (MTB) in patient samples. For proof of principle, we developed a multiplex PCR assay and report its validation with 500 clinical cultures, positive for Mycobacteria. The analysis revealed 98.9% sensitivity, 96.1% specificity, positive and negative predictive values of 98.6% and 96.8%, respectively. These results underscore the diagnostic value of the sRNA gene as diagnostic marker for the specific detection of MTB in clinical samples. Its successful application and the general ease of PCR-based detection compared to standard bacterial culture techniques might be the first step towards 'point-of-care' diagnostics of Mycobacteria. To the best of our knowledge, this is the first time for the design of diagnostic applications based on sRNA genes, in Mycobacteria.

Matched MeSH terms: DNA, Complementary/genetics
Fulltext Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria

Junaid QO, Khaw LT, Mahmud R, Ong KC, Lau YL, Borade PU, et al.

Parasite, 2017;24:38.
PMID: 29034874 DOI: 10.1051/parasite/2017040

BACKGROUND: As the quest to eradicate malaria continues, there remains a need to gain further understanding of the disease, particularly with regard to pathogenesis. This is facilitated, apart from in vitro and clinical studies, mainly via in vivo mouse model studies. However, there are few studies that have used gerbils (Meriones unguiculatus) as animal models. Thus, this study is aimed at characterizing the effects of Plasmodium berghei ANKA (PbA) infection in gerbils, as well as the underlying pathogenesis.
METHODS: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology.
RESULTS: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization.
CONCLUSION: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis.

Matched MeSH terms: DNA, Complementary/genetics
Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs

Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH

Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
PMID: 9565688

Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.

Matched MeSH terms: DNA, Complementary/genetics
Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase

Huang CH, Liew LM, Mah KW, Kuo IC, Lee BW, Chua KY

Clin Exp Allergy, 2006 Mar;36(3):369-76.
PMID: 16499649

Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems.

Matched MeSH terms: DNA, Complementary/genetics
Fulltext Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD)

Hu T, Zheng Y, Zhang Y, Li G, Qiu W, Yu J, et al.

BMC Microbiol, 2012;12:305.
PMID: 23268691 DOI: 10.1186/1471-2180-12-305

The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.

Matched MeSH terms: DNA, Complementary/genetics*
Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12

Hashim NH, Bharudin I, Nguong DL, Higa S, Bakar FD, Nathan S, et al.

Extremophiles, 2013 Jan;17(1):63-73.
PMID: 23132550 DOI: 10.1007/s00792-012-0494-4

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.

Matched MeSH terms: DNA, Complementary/genetics
Genetic characterization of Nipah virus, Bangladesh, 2004

Harcourt BH, Lowe L, Tamin A, Liu X, Bankamp B, Bowden N, et al.

Emerg Infect Dis, 2005 Oct;11(10):1594-7.
PMID: 16318702

Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks.

Matched MeSH terms: DNA, Complementary/genetics
Fulltext Efficient oil palm total RNA extraction with a total RNA extraction kit

Habib SH, Saud HM, Kausar H

Genet. Mol. Res., 2014;13(2):2359-67.
PMID: 24781991 DOI: 10.4238/2014.April.3.8

Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem.

Matched MeSH terms: DNA, Complementary/genetics*
Development of a comparative genetic linkage map for Armigeres subalbatus using Aedes aegypti RFLP markers

Ferdig MT, Taft AS, Severson DW, Christensen BM

Genome Res, 1998 Jan;8(1):41-7.
PMID: 9445486

One of the causative agents of lympahtic filariasis is the nematode parasite Brugia malayi that requires a competent mosquito vector for its development and transmission. Armigeres subalbatus mosquitoes rapidly destroy invading B. malayi microfilariae via a defense response known as melanotic encapsulation. We have constructed a genetic linkage map for this mosquito species using RFLP markers from Aedes aegypti. This heterologous approach was possible because of the conserved nature of the coding sequences used as markers and provided an experimental framework to evaluate the hypothesis that linkage and gene order are conserved between these mosquito species. Of the 56 Ae. aegypti markers tested, 77% hybridize to genomic DNA digests of Ar. subalbatus under stringent conditions, with 53% of these demonstrating strain-specific polymorphisms. Twenty-six Ae. aegypti markers have been mapped using an F2- segregating Ar. subalbatus population derived from a cross of strains originating in Japan and Malaysia. Linear order of these marker loci is highly conserved between the two species. Only 1 of these markers, LF92, was not linked in the manner predicted by the Ae. aegypti map. In addition, the autosomal sex-determination locus that occurs in linkage group 1 in Ae. aegypti resides in group 3 in Ar. subalbatus. The Ar. subalbatus map provides a basic genetic context that can be utilized in further genetic studies to clarify the genetic basis of parasite resistance in this mosquito and is a necessary precursor to the identification of genome regions that carry genes that determine the encapsulation phenotype. [The composite map and sequence database information for Ae. aegypti markers can be retrieved directly from the Ae. aegypti Genome Database through the World Wide Web: http://klab.agsci.colostate.edu.]

Matched MeSH terms: DNA, Complementary/genetics

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