William S. Modi

Interleukin-2 receptor γ chain mutation results in X-linked severe combined immunodeficiency in humans

The interleukin-2 (IL-2) receptor gamma chain (IL-2R gamma) is a component of high and intermediate affinity IL-2 receptors that is required to achieve full ligand binding affinity and internalization. We have localized the IL-2R gamma gene to human chromosome Xq13. Genetic linkage analysis indicates that the IL-2R gamma gene and the locus for X-linked severe combined immunodeficiency (XSCID) appear to be at the same position. Moreover, we demonstrate that each of three unrelated patients with XSCID has a different mutation in his IL-2R gamma gene resulting in a different premature stop codon and predicted C-terminal truncation. These data establish that XSCID is associated with mutations of the IL-2R gamma gene product. Since XSCID is characterized by absent or markedly reduced numbers of T cells, our findings imply that IL-2R gamma plays a vital role in thymic maturation of T cells. These results also have important implications for prenatal and postnatal diagnosis, carrier female detection, and gene therapy for XSCID.

Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat

The mitochondrial DNA of plant and animal cells is a transcriptionally active genome that traces its origins to a symbiotic infection of eucaryotic cells by bacterial progenitors. As prescribed by the Serial Endosymbiosis Theory, symbiotic organelles have gradually transferred their genes to the eucaryotic genome, producing a functional interaction of nuclear and mitochondrial genes in organelle function. We report here a recent remarkable transposition of 7.9 kb of a typically 17.0-kb mitochondrial genome to a specific nuclear chromosomal position in the domestic cat. The integrated segment has subsequently become amplified 38–76 times and now occurs as a tandem repeat macrosatellite with multiple-length alleles resolved by pulse-field gel electrophoresis (PFGE) segregating in cat populations. Sequence determination of the nuclear mitochondrial DNA segment, Numt, revealed a d(CA)-rich 8-bp motif [ACACACGT] repeated imperfectly five times at the deletion junction that is a likely target for recombination. The extent and pattern of sequence divergence of Numt genes from the cytoplasmic mtDNA homologues plus the occurrence of Numt in other species of the family Felidae allowed an estimate for the origins of Numt at 1.8–2.0 million years ago in an ancestor of four modern species in the genus Felis. Numt genes do not function in cats; rather, the locus combines properties of nuclear minisatellites and pseudogenes. These observations provide an empirical glimpse of historic genomic events that may parallel the accommodation of organelles in eucaryotes.

cDNA cloning and characterization of the human interleukin 13 receptor alpha chain.

We have cloned cDNAs corresponding to the human interleukin 13 receptor α chain (IL-13Rα). The protein has 76% homology to murine IL-13Rα, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13Rα; however, the combination of both IL-13Rα and IL-4Rα resulted in substantial binding activity, with a Kd of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13Rα serves as an alternative accessory protein to the common cytokine receptor γ chain (γc) for IL-4 signaling, it could not replace the function of γc in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13Rα and γc are similar, and like γc, IL-13Rα is located on chromosome X.

Modulating influence on HIV/AIDS by interacting RANTES gene variants

RANTES (regulated on activation normal T cell expressed and secreted), a ligand for the CC chemokine receptor 5, potently inhibits HIV-1 replication in vitro. We tested the influence of four RANTES single nucleotide polymorphism (SNP) variants and their haplotypes on HIV-1 infection and AIDS progression in five AIDS cohorts. Three SNPs in the RANTES gene region on chromosome 17 (403A in the promoter, In1.1C in the first intron, and 3′222C in the 3′ untranslated region) are associated with increased frequency of HIV-1 infection. The common In1.1C SNP allele is nested within an intronic regulatory sequence element that exhibits differential allele binding to nuclear proteins and a down-regulation of gene transcription. The In1.1C allele or haplotypes that include In1.1C display a strong dominant association with rapid progression to AIDS among HIV-1-infected individuals in African-American, European-American, and combined cohorts. The principal RANTES SNP genetic influence on AIDS progression derives from the down-regulating RANTES In1.1C allele, although linkage disequilibrium with adjoining RANTES SNPs including a weaker up-regulating RANTES promoter allele (−28G), can modify the observed epidemiological patterns. The In1.1C-bearing genotypes account for 37% of the attributable risk for rapid progression among African Americans and may also be an important influence on AIDS progression in Africa. The diminished transcription of RANTES afforded by the In1.1C regulatory allele is consistent with increased HIV-1 spread in vivo, leading to accelerated progression to AIDS.

Localization of the von Hippel-Lindau disease gene to a small region of chromosome 3

We studied 25 families with von Hippel-Lindau disease (VHL) to locate VHL more precisely on chromosome 3. We found that VHL was linked to RAF1, confirming previous observations, and to two polymorphic DNA markers, D3S18 and D3S191. Multipoint linkage analysis indicated that the most likely location for VHL was in the interval between RAF1 and D3S18. D3S18 was located at 3p26. Genetic heterogeneity was not detected in this panel of von Hippel-Lindau disease families. The polymorphic markers RAF1, D3S18, and D3S191 should be useful in identifying asymptomatic gene carriers in VHL families and in guiding efforts at gene isolation.

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily

SummaryMonocyte-derived neutrophil chemotactic factor (MDNCF/IL-8, suggested gene symbol IL8) is a cytokine that chemoattracts and activates neutrophils. Using a panel of human-rodent cell hybrids that preferentially segregate human chromosomes and in situ hybridization, the MDNCF/IL-8 gene was placed on the human gene map at position 4q12-q21. This is the same location where at least three other members (platelet factor 4, melanoma growth stimulatory activity, and interferon-γ induced factor) of the platelet factor 4 gene superfamily reside. In addition, a restriction fragment length polymorphism was identified using MDNCF as a probe in screening genomic DNA digested with HindIII from unrelated individuals.

The value of avian genomics to the conservation of wildlife.

BackgroundGenomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution.Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care).In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes.ResultsGenotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current populations genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology.Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one.ConclusionMining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease.In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

Evolutionary histories of highly repeated DNA families among the Artiodactyla (Mammalia).

Six highly repeated DNA families were analyzed using Southern blotting and fluorescence in situ hybridization in a comparative study of 46 species of artiodactyls belonging to seven of the eight extant taxonomic families. Two of the repeats, the dispersed bovine-Pst family and the localized 1.715 component, were found to have the broadest taxonomic distributions, being present in all pecoran ruminants (Giraffidae, Cervidae, Antilocapridae, and Bovidae), indicating that these repeats may be 25–40 million years old. Different 1.715 restriction patterns were observed in different taxonomic families, indicating that independent concerted evolution events have homogenized different motifs in different lineages. The other four satellite arrays were restricted to the Bovini and sometimes to the related Boselaphini and Tragelaphini. Results reveal that among the two compound satellites studied, the two components of the 1.711 a originated simultaneously, whereas the two components of the 1.711b originated at two different historical times, perhaps as many as 15 million years apart. Systematic conclusions support the monophyly of the infraorder Pecora, the monophyly of the subfamily Bovinae (containing the Boselaphini, Bovini, and Tragelaphini), an inability to resolve any interrelationships among the other tribes of bovids, paraphyly of the genusBos with respect toBison, and a lack of molecular variation among two morphologically and ecologically distinct subspecies of African buffaloes (Syncerus caffer cafer andS. c. nanus). Cytogenetically, a reduction in diploid chromosome numbers through centric fusion in derived karyotypes is accompanied by a loss of centromeric satellite DNA. The nilgai karyotype contains an apparent dicentric chromosome as evidenced by the sites of 1.715 hybridization. Telomeric sequences have been translocated to the centromeres without concomitant chromosomal rearrangement in Thompsons gazelle.

Morphological Variability and Asymmetry in the Cheetah (Acinonyx jubatus), a Genetically Uniform Species

The African cheetah (Acinonyx jubatus) is an unusual species because of its extremely low amount of biochemical genetic variation. A comparative analysis of morphological variation of 16 cranial characters from four species of Felidae (ocelot, Leopardus pardalus; margay, L. wiedii; leopard, Panthera pardus; and cheetah) was undertaken to evaluate the consequence of biochemical monomorphism on morphological variation. The species were selected because the cheetah has been shown previously to possess extremely low amounts of biochemical genetic variation as opposed to the other three species which retain comparatively high levels of allozyme heterozygosity. The cheetah sample showed dramatically greater fluctuating asymmetry but was not outstanding in morphological variability. Elevated levels of fluctuating asymmetry have been interpreted as a reflection of developmental instability, which is a common consequence of inbreeding. The inverse correlation of genetic variation and developmental stability (homeostasis) observed here fulfills prior expectations and further emphasizes the genetic invariability of the cheetah species.

Polymorphisms of the human IFNG gene noncoding regions

Abstract Interferon gamma (IFN-gamma) is a multifunctional cytokine that is essential in the development of Th1 cells and in cellular responses to a variety of intracellular pathogens including human immunodeficiency virus (HIV-1). We screened genomic DNA samples from a predominately Caucasian male population of HIV-infected and healthy donors for polymorphisms in the human IFNG gene from –777 to +5608 by single-stranded conformational polymorphism. Surprisingly, the proximal promoter (–777 to transcription start) is invariant as no polymorphisms were found in over 100 samples tested. However, further screening revealed polymorphisms in other regions of the gene including a single base insertion in a poly-T tract in the first intron, three single base pair substitutions in the third intron, and another single base pair substitution in the 3′ untranslated region (UTR). Electrophoretic mobility shift assay was used to investigate whether these variants have altered DNA-binding abilities, since intronic enhancer elements have been reported for the IFNG gene. Oligonucleotides constructed for two third intron variants showed no difference in DNA-binding abilities as compared with wild-type sequences. However, the 3′UTR variant showed the formation of unique DNA-binding complexes to radiolabeled oligonucleotide probes as compared with the wild-type sequence. The influence of a CA-repeat microsatellite on AIDS disease progression in HIV-1 seroconverters was tested by a Cox proportional hazards model. There is no evidence of an association between alleles and infection with HIV-1 or progression to AIDS. We report an invariant proximal human IFNG promoter and the existence of multiple intronic variants and a potentially functional 3′UTR polymorphism.