Identification of candidate genes regulating HDL cholesterol using a chromosomal region expression array

To identify candidate genes encoding QTLs in baboons, we have developed a novel strategy that integrates comparative mapping, bioinformatics, and expression arrays. A genome-wide scan, performed previously on pedigreed baboons to localize QTLs for phenotypes that are known risk factors for atherosclerosis, revealed a QTL on chromosome 18q that influences high-density lipoprotein cholesterol (HDL-C) phenotypes. After ruling out the only two biologically relevant positional candidate genes in this chromosomal region, we combined information from baboon pedigrees and HDL-C phenotypes with a baboon microsatellite marker map, human microsatellite marker maps, and human genome maps to develop a chromosomal region expression array (CREA). The CREA was screened with heterologous liver cDNA from sib-pairs of contrasting HDL-C phenotypes on two different diets, and genes were prioritized for further study by expression profiles. Analysis of gene expression in this restricted chromosomal region, combined with HDL-C phenotypic information, yielded a list of candidate genes for the QTL regulating HDL-C in baboons. Our data demonstrate the power of this strategy for identifying candidate genes encoding QTLs for multigenic traits. This strategy is applicable to many species that serve as models for human diseases and can even be used with human subjects.     

Identification of a self-association region within the SCA1 gene product, ataxin-1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract within the SCA1 gene product, ataxin-1. Expansion of this tract is believed to result in a gain of function by the mutant protein, perhaps through altered self-associations or interactions with other cellular proteins. We have used the yeast two hybrid system to determine if ataxin-1 is capable of multimerization. This analysis revealed that ataxin-1 does have the ability to self-associate, however, this association does not appear to be influenced by expansion of the polyglutamine tract. Consistent with this finding, deletion analysis excluded the involvement of the polyglutamine tract in ataxin- 1 self-association, and instead localized the multimerization region to amino acids 495-605 of the wild type protein. These results, while identifying an ataxin-1 self-interaction region, fail to support a proposed model of polar-zipper mediated multimerization involving the ataxin-1 polyglutamine tract.      

Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF are the catalytic components of the toxin, whereas PA is the receptor-binding component. To identify residues of PA that are involved in interaction with the cellular receptor, two solvent-exposed loops of domain 4 of PA (amino acids [aa] 679 to 693 and 704 to 723) were mutagenized, and the altered proteins purified and tested for toxicity in the presence of LF. In addition to the intended substitutions, novel mutations were introduced by errors that occurred during PCR. Substitutions within the large loop (aa 704 to 723) had no effect on PA activity. A mutated protein, LST-35, with three substitutions in the small loop (aa 679 to 693), bound weakly to the receptor and was nontoxic. A mutated protein, LST-8, with changes in three separate regions did not bind to receptor and was nontoxic. Toxicity was greatly decreased by truncation of the C-terminal 3 to 5 aa, but not by their substitution with nonnative residues or the extension of the terminus with nonnative sequences. Comparison of the 28 mutant proteins described here showed that the large loop (aa 704 to 722) is not involved in receptor binding, whereas residues in and near the small loop (aa 679 to 693) play an important role in receptor interaction. Other regions of domain 4, in particular residues at the extreme C terminus, appear to play a role in stabilizing a conformation needed for receptor-binding activity.     

Novel mutations in the duplicated region of PKD1 gene

Autosomal dominant polycystic kidney disease (ADPKD) exhibits a genetically heterogeneous transmission involving at least three different genes. PKD1 gene linked mutations are responsible for the disease in about 85% of ADPKD cases. The search for mutations is a very important step in understanding the molecular mechanisms underlying ADPKD. We undertook this study using denaturing gradient gel electrophoresis (DGGE), after a stage of long range PCR, to scan for mutations in the duplicated region of the PKD1 gene in French ADPKD families. This allowed us to identify eight novel mutations and several polymorphisms: among the mutations, three are nonsense mutations, two are deletions in the coding sequence leading to frameshift mutations, one is a splice mutation and two are highly probable missense mutations. In this paper, we also provide a review of the mutations reported so far which are widespread throughout the gene. Although no clear hot spot for mutation is apparent, we will focus on some clustering observed.     

Identification of a novel DNase I hypersensitive site within the far upstream region of the human HLA-DRA gene

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.     

A sensitive method for detecting variation in copy numbers of duplicated genes

Gene duplications are common in the vertebrate genome, and duplicated loci often show a variation in copy number that may have important phenotypic effects. Here we describe a powerful method for quantification of duplicated copies based on pyrosequencing. A reliable quantification was obtained by amplification of the duplication break-point and a corresponding nonduplicated sequence in a competitive PCR assay. A comparison with an independent method for quantification based on the Invader technology revealed an excellent correlation between the two methods. The pyrosequencing-based method was evaluated by analyzing variation in copy number at the duplicated KIT/Dominant white locus in pigs. We were able to distinguish haplotypes at this locus by combining the duplication breakpoint test with a diagnostic test for a functionally important splice mutation in the duplicated gene. An extensive allelic variation, including the presence of a new allele carrying a single KIT copy expected to encode a truncated KIT receptor, was revealed when analyzing white pigs from commercial lines.     

Multiple replication origins within the inverted repeat region of the Plasmodium falciparum apicoplast genome are differentially activated

The 35 kb circular genome (plDNA) of the Plasmodium falciparum apicoplast replicates by the bidirectional ori/D-loop mechanism. PlDNA replication was previously shown to initiate within the inverted repeat (IR) region of the apicoplast genome [Williamson DH, Preiser PR, Moore PW, McCready S, Strath M, Wilson RJM (Iain). The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms. Mol Microbiol 2002;45:533-42; Singh D, Chaubey S, Habib S. Replication of the Plasmodium falciparum apicoplast DNA initiates within the inverted repeat region. Mol Biochem Parasitol 2003;126:9-14.] and the presence of at least two ori within each segment of the IR was postulated. Using 5' end-labelled nascent DNA as probe, we now demonstrate the utilization of several putative ori located within the IR for plDNA replication. Quantitation of signals obtained for different segments of the IR as well as determination of the number of molecules emanating from two ori regions by competitive PCR analysis indicated differential strengths of ori during plDNA replication prior to schizogony.     

Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.     

The fate of duplicated major histocompatibility complex class Ia genes in a dodecaploid amphibian,Xenopus ruwenzoriensis

The dodecaploid anuran amphibian Xenopus ruwenzoriensis represents the only polyploid species of Xenopus in which the full silencing of the extra copies of the major histocompatibility complex (MHC) has not occurred. Xenopus ruwenzoriensis is a recent polyploid that has evolved within one of the two tetraploid groups of Xenopus through allopolyploidization. Family studies of its MHC haplotype suggested a polysomic inheritance of the MHC class I and II genes. Four class Ia bands can be detected per individual in Southern blot analysis and, similarly, four different cDNA sequences are expressed per individual. The Xenopus class Ia sequences we analyzed belong to only one of the old class I lineages and show a homogenization of their alpha3 domain sequences. This homogenization occurred after speciation within the Xenopus ruwenzoriensis species, either due to gene conversion or inter-alleles/loci recombination.A re-evaluation of the polymorphism of class Ia in Xenopus, by looking at the rate of non-synonymous versus synonymous substitutions, suggests that Xenopus MHC class Ia genes are not under strong overdominant selection. This is a rare situation among vertebrates. The observed polymorphism is most likely due to the interlocus genetic exchanges related to the peculiar mode of speciation of the genus.     

The fate of duplicated major histocompatibility complex class Ia genes in a dodecaploid amphibian,Xenopus ruwenzoriensis

The dodecaploid anuran amphibian Xenopus ruwenzoriensis represents the only polyploid species of Xenopus in which the full silencing of the extra copies of the major histocompatibility complex (MHC) has not occurred. Xenopus ruwenzoriensis is a recent polyploid that has evolved within one of the two tetraploid groups of Xenopus through allopolyploidization. Family studies of its MHC haplotype suggested a polysomic inheritance of the MHC class I and II genes. Four class Ia bands can be detected per individual in Southern blot analysis and, similarly, four different cDNA sequences are expressed per individual. The Xenopus class Ia sequences we analyzed belong to only one of the old class I lineages and show a homogenization of their alpha3 domain sequences. This homogenization occurred after speciation within the Xenopus ruwenzoriensis species, either due to gene conversion or inter-alleles/loci recombination.A re-evaluation of the polymorphism of class Ia in Xenopus, by looking at the rate of non-synonymous versus synonymous substitutions, suggests that Xenopus MHC class Ia genes are not under strong overdominant selection. This is a rare situation among vertebrates. The observed polymorphism is most likely due to the interlocus genetic exchanges related to the peculiar mode of speciation of the genus.     

Identification of seven genes in the major histocompatibility complex class I region of the zebrafish

Physical linkage of genes whose products are involved in similar physiological pathways may have functional significance. The identification of conserved gene linkage in distantly related organisms can therefore strengthen the hypothesis of selection acting towards keeping genes on a chromosome. We used the cDNA selection technique and the polymerase chain reaction (PCR) with generic primers for the identification of new genes on the genomic clones bearing the major histocompatibility complex (Mhc) class I genes of the zebrafish (Danio rerio). We found six new genes (BING1, DAXX, TAPBP, KNSL2, TAP2B and KE6) whose orthologues are known to be linked to the Mhc class II region in humans and mice. In addition, a new zebrafish Mhc class I gene, termed Dare-UFA, was detected. By contrast, a search for the human leucocyte antigen (HLA)-linked BING3, KE3 and SACM2L genes revealed that these loci are not located on the class I clones of the zebrafish. The zebrafish class I region contains repetitive elements with similarity to the DANA, SATA and LINE repeats, as well as Tc1 transposable elements. Our findings indicate a high degree of linkage conservation between the zebrafish class I and the mammalian class II regions.     

A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC

In the polyoma virus (Py) transformed 3B rat cell line the Pyoncogene and adjacent cellular DNA are amplified in arrays ofvery large inverted duplications. A region of the 3B amplifiedDNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome(YAC) vector, designated y3B01. Analysis of the y3B01 clonedinsert revealed it contained a large inverted duplicated DNAregion which was approximately 400 kb in size (two palindromicarms of about 200 kb). At least 420 kb of the 550 kb YAC inserthas been identified as being derived from the 3B amplified DNAand the amplicon in 3B cells is at least 220 kb in size. NoDNA instability of the y3B01 YAC clone was detected. The y3B01DNA replicated as efficiently as yeast chromosomes and was structurallystable in yeast cells during more than 30 cell divisions. Comparisonof the restriction endonuclease maps of the inverted duplicatedregion of the y3B01 DNA insert and the amplified 3B genomicDNA did not reveal any gross differences suggesting that norearrangements had occurred during or after the cloning intothe YAC vector. These results suggest that large inverted duplications,which can show instability in prokaryotic cloning systems, canbe stably cloned and maintained in YAC vectors in yeast.