Members Of Multigene Family Research Articles

Association of mitochondrial D-loop mutations with GSTM1 and GSTT1 polymorphisms in oral carcinoma: A case control study from Northeast India

Mitochondrial dysfunction is a hallmark of cancer cells. Tobacco consumption in various forms is one of the major risk factors for the development of oral squamous cell carcinoma which makes the mitochondrial DNA susceptible to damage by reactive oxygen species. The GSTT1 and GSTM1 members of the glutathione S-transferase multigene family are candidate carcinogen metabolizing genes. Here we determined the hot spot mutations in the D-loop region and revealing correlation if any, with clinical parameters, along with analysing the genetic polymorphism of GSTT1 and GSTM1 and its susceptibility towards oral cancer. To determine the hot spot mutations 25 matched tissue samples of OSCC patients with 25 control subjects were used for PCR and direct sequencing. Analysis for GSTM1 and GSTT1 gene polymorphism was done by multiplex PCR. Several mutations were found within the D-loop region among which mutations at nt146, nt152 and nt196 are found to be hot spot (P<0.0001, P<0.0001 and P<0.001 respectively). A significant association was found between the numbers of D-loop mutation and GSTM1 (OR=2.03; 95% CI, 1.04-3.96, P=0.003), GSTT1 (OR=1.73; 95% CI, 1.10-2.71, P=0.0027) null genotypes respectively. We observed a significant correlation between the increased number of D-loop mutations with the advancement in tumour stage of the patients (P=0.009, r=0.48). The association of null genotypes and mutations can be used as a possible biomarker for early detection and preventive measure of oral cancer for those habituated to tobacco consumption.

Identification and characterization of new major sperm protein genes from Oesophagostomum dentatum and Oesophagostomum quadrispinulatum from pigs in China

The present study identified and characterized new major sperm protein (MSP) genes from the two nodule worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum collected from pigs in China. Total genomic DNA was extracted individually from 10 male nematode samples representing O. dentatum, and 4 male nematode samples representing O. quadrispinulatum. A pair of primers (OMSP1F/MSP1R) was designed based on the MSP gene sequences of Ascaris suum and O. dentatum available in GenBank, and used to amplify the MSP genes from the two porcine nodule worms. The PCR products were purified and subsequently cloned into pGEM-T Easy vector. Recombinants were identified by PCR and sequenced. Sequence analysis revealed that there were two different types of MSP sequences in O. dentatum and O. quadrispinulatum, one contained intron, and the other did not. The lengths of the MSP sequences containing introns were 433 bp or 439 bp in O. dentatum, and 436 bp, 439 bp or 446 bp in O. quadrispinulatum, containing 1 or 2 introns. Five and three new members of the MSP multigene family were identified in O. dentatum and O. quadrispinulatum in this study, respectively. The MSP sequences without introns were 381 bp in length, and can be deduced into 126 amino acids. The sequences of MSP genes containing introns seem to be more conserved than those without introns. The identities of deduced amino acid sequences of the MSP genes containing introns were 96.0-100% within and between the two nodule worms, and were 81.1-93.7% compared with other published MSP sequences of the representative nematodes. The present study identified new MSP genes with introns from O. dentatum and O. quadrispinulatum for the first time. The identification and characterization of newly described MSP genes from O. dentatum and O. quadrispinulatum have implications for further studies of molecular biology and reproduction control of Oesophagostomum spp.

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family

Rapid antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein with subunits encoded by two branches of the ves multigene family. The ves1α and ves1β gene pair encoding VESA1a and 1b, respectively, are transcribed in a monoparalogous manner from a single locus of active ves transcription (LAT), just one of many quasi-palindromic ves loci. To determine whether this organization plays a role in transcriptional regulation, chromatin structure was first assessed. Limited treatment of isolated nuclei with micrococcal nuclease to assay nucleosomal patterning revealed a periodicity of 156–159bp in both bulk chromatin and specific gene coding regions. This pattern also was maintained in the intergenic regions (IGr) of non-transcribed ves genes. In contrast, the LAT IGr adopts a unique pattern, yielding an apparent cluster of five closely-spaced hypersensitive sites flanked by regions of reduced nucleosomal occupancy. ves loci fall into three patterns of overall sensitivity to micrococcal nuclease or DNase I digestion, with only the LAT being consistently very sensitive. Non-transcribed ves genes are inconsistent in their sensitivity to the two enzymatic probes. Non-linear DNA structure in chromatin was investigated to determine whether unique structure arising as a result of the quasi-palindromic nature of the LAT may effect transcriptional control. The in vitro capacity of ves IGr sequences to adopt stable higher-order DNA structure is demonstrated here, but the presence of such structure in vivo was not supported. Based upon these results a working model is proposed for the chromatin structural remodeling responsible for the sequential expression of ves multigene family members from divergently-organized loci.

Backbone and side-chain 1H, 15N and 13C assignments of mouse peptide ESP4

A peptide or a small protein released from an exocrine gland or in urine is utilized as a chemosignal that elicits social or reproductive behavior in mice. Recently, we identified the male-specific peptide, exocrine gland-secreting peptide 1 (ESP1), in mouse tear fluids that enhanced female sexual receptive behavior, and determined the three dimensional structure. ESP1 appears to be a member of multigene family that consists of 38 genes in mice, which we call the ESP family. ESP4, a member of the ESP family, is expressed in various exocrine glands, and shows the highest sequence similarity with ESP1. Here, we report the NMR assignments of ESP4 which provides a basis for NMR analyses of this protein. Our results will give insight into structural relationships within the ESP family.

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.

Reflex™: a novel method to sequence barcoded long-range PCR products in a pooled population of hundreds of DNA samples

We are developing novel methods to simultaneously analyze candidate genes or regions from multiple samples in a single experiment rapidly and cost-effectively. One method, we call Reflex, provides an elegant approach to sample preparation for long-range PCR (LR-PCR) amplicons. Current methods use LR-PCR for target enrichment then use random fragmentation of each sample followed by ligation of DNA barcodes before sequencing: this approach is expensive and labour intensive. In the Reflex workflow, we perform LR-PCR on genomic targets using primers that also add a DNA barcode to the LR-PCR product. This allows us to then produce pools of the LR-PCR products of multiple, typically 384, samples. The equimolar pooled population of 384 LR-PCR products are then processed to generate tiled 'daughter' Reflex PCR products across the target, each carrying a copy of the cognate DNA barcode. This is achieved by an intramolecular fold-back between two inverted Reflex sequences, followed by polymerase extension, so copying the DNA barcode. The Reflex daughter PCR products are then equimolar pooled for sequencing where the DNA barcode allows identification of the originating DNA sample within the population pool. We have developed Reflex workflows that are compatible with the Illumina, Ion Torrent and Roche-454 next-generation sequencing platforms and can index multiple pooled populations to sequence thousands of DNA samples in a single run. The workflow is robust and can be performed quickly and cheaply with small amounts of input DNA and with high specificity to discriminate between members of multigene families.

The repertoire diversity of the Plasmodium falciparum stevor multigene family in complicated and uncomplicated malaria in India

Background The deep vascular sequestration of parasitized erythrocytes is a central pathological event in falciparum malaria. Variant surface antigens are encoded mainly by three multi-copy gene families, namely, var, stevor ,a ndrifin. Var is one of the most important families that plays a crucial role in antigenic variation and immune evasion. Clinical and epidemiological studies have shown that severe or complicated malaria is manifested in a limited number of patients. This indicates that a subset of these multigene families could be determinants in the manifestation of different malaria phenotypes. Recent studies have indicated the possible role of stevor (sub-telomeric variable open read), a multi-gene family, in erythrocyte invasion, antigenic variation and host cell modification, of infected erythrocytes. In this study, we describe the repertoire and diversity of members of the stevor multigene family in patients with complicated and uncomplicated malaria in India.

Allelic variation of GSTM1 and GSTT1 genes in Haryana population

The glutathione S-transferase mu (GSTM1) and theta (GSTT1) members of the GST multigene family are polymorphic in human populations, and homozygous deletions or null genotypes of GSTT1 and GSTM1 genes have been reported in different populations. Epidemiological studies suggest that individuals who are homozygous null at the GSTM1 or GSTT1 loci may have an increased risk of cancer; therefore, the distribution of these genotypes in the human population is of great interest. The aim of our study was to investigate the allelic variations of GSTM1 and GSTT1 genotypes in 308 healthy, unrelated individuals from the state of Haryana in India. The participants were genotyped for the presence of GSTM1 and GSTT1 genes using the multiplex polymerase chain reaction (PCR) technique. On the basis of the results from our findings, we found that, in the geographic region of our study (India), 40.58% of individuals are carriers of the GSTM1 0/0 (null) genotype, whereas 32.79% of individuals have the GSTT1 0/0 (null) genotype. This study contributes significant information on the variability of GSTT1 and GSTM1 gene polymorphisms in the worldwide population and could increase knowledge about the relationship between ethnicity and the prevalence of certain diseases.

The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60–80 % of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.

Transcriptional analysis of physiological pathways in a generalist herbivore: responses to different host plants and plant structures by the cotton bollworm, Helicoverpa armigera

AbstractThe generalist cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), can consume host plants in more than 40 families, and often utilizes several tissues of a single plant. It is believed that generalists owe their success to the deployment of various members of multigene families of detoxification and digestive enzymes, a strategy that may also be responsible for rapid evolution of insecticide resistance. However, studies of generalist adaptations have been limited to specific genes or gene families, and an overview of how these adaptations are orchestrated at the transcriptional level is lacking. We used Drosophila melanogaster Meigen gene homology to H. armigera‐expressed sequence tags to identify key groups of genes and pathways differentially regulated in the gut of fifth instars after 2 days of feeding on a variety of food sources. A series of microarray hybridizations was performed following two alternating loop designs, one comparing the gut gene expression upon feeding on various hosts (cotton, bean, tobacco, and chickpea) and two artificial diets (pinto bean and wheat germ‐based), whereas the second design compared the gut expression toward feeding on various plant structures within cotton (leaf, square, and boll). The transcriptional responses toward bean and tobacco feeding treatments were more closely related in comparison with the rest of the diets, whereas the gene expression profiles toward cotton leaf and square‐feeding were highly similar. We furthermore found significant changes in several pathways not directly responsible for detoxification mechanisms. Genes involved in primary and secondary metabolism, environmental information processing, and cellular processes were found to be differentially expressed. In addition, regulation of xenobiotic metabolism and the extracellular matrix‐receptor pathways appeared differentially regulated across feeding treatments. Three cytochrome P450 genes – CYP6AE17, CYP6B6, and CYP9A17 – grouped as part of a xenobiotic metabolism pathway, were up‐regulated in the bean‐feeding treatment, and down‐regulated in both tobacco and cotton‐feeding treatments. CYP4L11, CYP4L5, and CYP4S13 were differentially expressed upon feeding on different cotton plant structures. The present work provides host plant and plant structure‐specific transcriptional responses in a lepidopteran herbivore, including pathways and gene candidates for future studies of H. armigera physiology under a more integrative ecologically meaningful framework.

Brown midrib2 (Bmr2) encodes the major 4‐coumarate:coenzyme A ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench)

Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation.

The dimerization of glucagon‐like peptide‐2 MIMETIBODY™ is linked to leucine‐17 in the glucagon‐like peptide‐2 region

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY™ refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY™ scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY™ in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY™, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.

13 Gene Expression Pattern of Arabidopsis EXPB1, a Nonallergenic Homologue of Grass Group 1 Pollen Allergens

BackgroundGrass pollen allergy is one of the most common allergies worldwide. Group I allergens constitute the major allergenic component of grass pollen with more than 85% of grass pollen allergic patients showing IgE reactivity. These are highly immunologically cross-reactive glycoproteins specifically expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function.MethodsRT-PCR analysis, In situ hybridisation, Promoter-GFP construct design, plant transformation and analysis of transgenic plants.ResultsSequence comparison has identified a homologue (β-expansin clone At2g20750 or EXPB1) in Arabidopsis of the Cyn d 1 gene. The EXPB1 protein is 42% similar to the Cyn d 1 protein. This gene represents a member of a small multigene family in Arabidopsis. RT-PCR analysis showed expression only in floral not vegetative tissues. In situ hybridisation using 150 bp region of the 3′ UTR of the Arabidopsis gene as probe showed specific expression in mature Arabidopsis pollen. We further cloned the promoter region for the Arabidopsis EXPB1 and prepared and GFP fusion constructs. These constructs were then introduced in to Arabidopsis plants by floral dip method. GFP-promoter fusions showed high level of expression in tri-cellular pollen.ConclusionsOur study provides evidence that EXPB1, a non-allergenic homologue of grass group 1 pollen allergens, gene is expressed in mature pollen.

Characterization of Squamate Olfactory Receptor Genes and Their Transcripts by the High-Throughput Sequencing Approach

The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms.

Melanoma Antigen Encoding Gene-A3 Expression, Its Clinical Significance, and Its Risk of Developing Acute Myeloid Leukemia

BackgroundMelanoma antigen encoding gene A3 (MAGE-A3) gene, also called cancer/testis (CT) antigen, is a member of MAGE multigene family, which is located on the long arm of the X chromosome,...

Comparison of embryonic expression within multigene families using the flyexpress discovery platform reveals more spatial than temporal divergence

Overlaps in spatial patterns of gene expression are frequently an initial clue to genetic interactions during embryonic development. However, manual inspection of images requires considerable time and resources impeding the discovery of important interactions because tens of thousands of images exist. The FlyExpress discovery platform was developed to facilitate data-driven comparative analysis of expression pattern images from Drosophila embryos. An image-based search of the BDGP and Fly-FISH datasets conducted in FlyExpress yields fewer but more precise results than text-based searching when the specific goal is to find genes with overlapping expression patterns. We also provide an example of a FlyExpress contribution to scientific discovery: an analysis of gene expression patterns for multigene family members revealed that spatial divergence is far more frequent than temporal divergence, especially after the maternal to zygotic transition. This discovery provides a new clue to molecular mechanisms whereby duplicated genes acquire novel functions. The application of FlyExpress to understanding the process by which new genes acquire novel functions is just one of a myriad of ways in which it can contribute to our understanding of developmental and evolutionary biology. This resource has many other potential applications, limited only by the investigator's imagination.

Genomic survey of early responses to viruses in Atlantic salmon, Salmo salar L.

Viral diseases are one of the main problems and risk factors in aquaculture. At present diseases are diagnosed by detection of pathogens and clinical symptoms. Identification of genes involved in early responses to viruses is important for better knowledge of antiviral defence and development of diagnostic tools. The aim of this study was to search for gene markers common for viral infections in Atlantic salmon based on microarray analyses of a wide range of samples. Gene expression profiles from fish and cell cultures infected with different viruses and treated with the synthetic double-stranded RNA poly(I:C) were compared in order to identify virus responsive genes (VRG). The list of VRG defined in this study contained 117 genes with known or unidentified functions. Several genes, including the most highly ranked one (receptor transporting protein), had not been previously reported to be involved in antiviral defence. VRG were characterized by a rapid induction and low tissue specificity, and their expression levels were related to the viral load. Immunofluorescence analyses of proteins encoded by VRG in cardiac tissue of salmon with the viral disease cardiomyopathy syndrome (CMS) revealed a common expression pattern. In head kidney leukocytes VRG showed comparable or equal responses to CpG and poly(I:C), which mimic respectively bacterial DNA and viral RNA. Most VRG showed highly correlated expression with interferon-a (IFNa). Sequence comparison of salmon VRG with those from other species gave an understanding of the evolution of these genes, which showed a remarkably rapid sequence divergence in comparison with the entire proteome. VRG emerged both before and after separation of teleosts and tetrapods, and among genes found exclusively in fish species there were members of several multigene families: tripartite motif proteins, gig1- and gig2-like proteins. Several VRG, including genes with unknown functions and orthologs to mammalian RNA helicase RIG-I and chemokine C-X-C type 10, were present in cyprinid and salmonid fish but not in the phylogenetically advanced orders, suggesting that they have been lost in the evolution of Teleostei. Apparently, a number of genes involved in antiviral responses in salmon have acquired different functional roles in higher vertebrates.

Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease1,2. Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases3, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin4,5 and PrgJ6, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC47–11, but is widely assumed to play only an auxiliary role1,2, since NAIP5 is often dispensable for NLRC4 activation7,8. However, Naip5 is a member of a small multigene family12, raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP/NLRC4 inflammasomes.

Development of Apolipoprotein E–Deficient Mice

The first mouse carrying a deletion mutation in its apolipoprotein E (apoE) gene was born in our mouse colony in the summer of 1991. Our ongoing experiments to that point had indicated that this would happen, but the realization that the mouse in front of my eyes had inherited a mutation that we had designed and made in the laboratory filled me with awe. It did not occur to me immediately that this mouse would develop hypercholesterolemia and atherosclerosis and dictate the direction of my research for the next 20 years. I here briefly describe the background and my personal experience that led to the development of apoE-deficient mice as a model for human atherosclerosis. As a research scientist in the Department of Genetics at the University of Wisconsin, my main interest during the early 1980s was directed toward understanding how different types of DNA recombination contribute to the architecture and diversity of individual genomes within human populations and to DNA differences between humans and nonhuman primates. My studies clearly showed that new alleles and new genes are constantly being formed in the human genome by recombinational events, particularly between existing members of multigene families. There also appeared to be hot spots for recombination. By tracing a history of the human haptoglobin-gene cluster from the present-day structures in human populations and in other primates, I found that a single recombination event between related genes often produces much more drastic changes in the genome than those that result from simple point mutations.1 Whether this frequent yet natural occurrence of homologous recombination in the mammalian genome could be used to alter the genome of mammalian cells in culture in a preplanned fashion became a question of interest to several laboratories.2,3 An affirmative answer was first obtained in 1985, …

Antibodies directed against merozoite surface protein-6 are induced by natural exposure to Plasmodium falciparum in a low transmission environment

Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and there is currently no licensed vaccine that provides protection against infection or disease. Several P.falciparum vaccine targets have undergone early testing, but many more candidates remain with little data to support their development. Plasmodium falciparum Merozoite Surface Protein 6 (PfMSP6) is a candidate of particular interest because it is a member of the PfMSP3 multi-gene family, raising the possibility that vaccine-induced immune responses could cross-react across multiple family members. However, few immunoepidemiological studies of PfMSP6 have been carried out to measure domain-specific anti-PfMSP6 responses. This study investigated anti-PfMSP6 responses in P.falciparum-infected individuals from the Peruvian Amazon, using two different PfMSP6 N-terminal allele antigens and a single C-terminal domain antigen, and compared the responses with both PfMSP6 genotyping data and anti-PfMSP3 response data that had been previously generated for the same samples. Anti-PfMSP6 responses were detected despite the low transmission setting, but were less frequent and of considerably lower intensity than anti-PfMSP3 responses. There was a positive correlation between anti-PfMSP3 and PfMSP6 responses, suggesting that the possibility that PfMSP3 family antigens could induce cross-reactive responses requires further detailed investigation.