Localization of subtelomeric sequences of human chromosomes 1q, 11p, 13q, and 16q in the higher primates

Relative phylogenetic divergence of the members of the Pongidae family has been based on genetic evidence. The recent isolation of subtelomeric probes specific for human (HSA) chromosomes 1q, 11p, 13q, and 16q has prompted us to cross hybridize these to the chromosomes of the chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo pygmaeus, PPY) to search for their equivalent locations in the great apes. Hybridization signals to the 1q subtelomeric DNA sequence probe were observed at the termini of human (HSA) 1q, PTR 1q, GGO 1q, PPY 1q, while the fluorescent signals to the 11p subtelomeric DNA sequence probe were observed at the termini of HSA 11p, PTR 9p, GGO 9p, and PPY 8p. Fluorescent signals to the 13q subtelomeric DNA sequence probe were observed at the termini of HSA 13q, PTR 14q, GGO 14q, and PPY 14q, and positive signals to the 16p subtelomeric DNA sequence probe were observed at the termini of HSA 16q, PTR 18q, GGO 17q, and PPY 19q. These findings apparently suggest sequence homology of these DNA families in the ape chromosomes. Obviously, analogous subtelomeric sequences exist in apes' chromosomes that apparently have been conserved through the course of differentiation of the hominoid species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical Mapping of Human 7q and 14q Subtelomeric DNA Sequences in the Great Apes

Phylogenetic divergence of the members of the Pongidae familyhas been based on genetic evidence. The terminal repeat array(T₂AG₃) has lately been considered as an additional basis toanalyze genomes of highly related species. The recent isolationof subtelomeric DNA probes specific for human (HSA) chromosomes7q and 14q has prompted us to cross-hybridize them to the chromosomesof the chimpanzee (PTR), gorilla (GGO) and orangutan (PPY) tosearch for its equivalent locations in the great ape species.Both probes hybridized to the equivalent telomeric sites ofthe long (q) arms of all three great ape species. Hybridizationsignals to the 7q subtelomeric DNA sequence probe were observedat the telomeres of HSA 7q, PTR 6q, GGO 6q and PPY 10q, whilehybridization signals to the 14q subtelomeric DNA sequence probewere observed at the telomeres of HSA 14q, PTR 15q, GGO 18qand PPY 15q. No hybridization signals to the chromosome 7-specificalpha satellite DNA probe on the centromeric regions of theape chromosomes were observed. Our observations demonstratesequence homology of the subtelomeric repeat families D7S427and D14S308 in the ape chromosomes. An analogous number of subtelomericrepeat units exists in these chromosomes and has been preservedthrough the course of differentiation of the hominoid species.Our investigation also suggests a difference in the number ofalpha satellite DNA repeat units in the equivalent ape chromosomes,possibly derived from interchromosomal transfers and subsequentamplification of ancestral alpha satellite sequences.     

Comparative mapping of human claudin-1 (CLDN1) in great apes

The gene encoding claudin-1 (CLDN1) has been mapped to human chromosome 3 (HSA3; 3q28-->q29) using a radiation hybrid panel. Employing fluorescence in situ hybridization (FISH) we here show that a human P1-derived artificial chromosome (PAC) containing CLDN1 detects the orthologous sites in chromosomes of the great apes, chimpanzee, gorilla, and orangutan. Furthermore, the chromosomal position of CLDN1 was determined in mouse chromosomes by FISH. The position of fluorescent signals is confined to a single chromosomal site in both great apes and mouse and in each case maps to the chromosomal region that has conserved synteny with HSA3 (PTR2q28, GGO2q28, PPY2q38 and MMU16B1). Using a gene-specific probe our results are consistent with reports of the striking similarity of great ape and human genomes as illustrated previously by chromosome painting.     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed V_λ and V_κ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.     

Human Vκ immunoglobulin gene number: Implications for the origin of antibody diversity

To assess the relative contributions of germline versus somatically mutated genes in the human immune system, we have examined the size of the kappa light-chain variable region (V_κ) gene pool. Two cloned kappa subgroup 1 (V_κ1) gene probes detected the same family of 15 to 20 crosshybridizing restriction fragments in human DNA, whereas flanking region probes detected fewer hybridizing fragments. Most of the hybridizing bands represent single-copy genes, as judged by a “gene titration” experiment. Furthermore, the number of hybridization bands is a good estimate of the haploid gene number, since we observed little polymorphism of restriction sites in the V_κ locus of eight unrelated people. A cloned V_κ3 probe hybridized to essentially the same 15–20 genes in human DNA as the V_κ1 probes. These results strongly suggest that a discrete family of 15–20 genes constitutes a large proportion of the V genes from three of the four V_κ subgroups. The small number of V_κ genes in the human genome supports the idea that somatic mutation plays a major role in the origin of antibody diversity in man.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

Complexity,polymorphism, and connectivity of mouse V k gene families

To define the polymorphism and extent of the mouse immunoglobulin kappa (Igk) gene complex, we have analyzed restriction-enzyme digested genomic DNA from 33 inbred strains of mice with labeled DNA probes corresponding to 16 V _x protein groups (1 of them previously undescribed) and the J _k/C _K region (V, variable; J, joining; C, constant). These probes detected between 1 and 25 distinct restriction enzyme fragments (REF) that appeared in up to eight polymorphic patterns, thus defining eight mouse Jgk haplotypes. The investigated portion of the V _A repertoire was estimated to encompass between 60 and 120 discernable V _k gene-containing REFs. In contrast to mouse V _H gene families, several V _k gene families defined by these probes appeared to overlap. This observation has implications for V _k gene analyses by nucleic acid hybridization and raises the possibility that the V _A gene complex is a continuum of related sequences.Abbreviations used in this paper C constant - Ig immunoglobulin - J joining - REF restriction enzyme fragment - RFLP restriction fragment length polymorphism - V variable     

New insights into the evolution of chromosome 1

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.     

The genomic sequence for Prader-Willi/Angelman syndromes' loci of human is apparently conserved in the great apes

Chromosomal changes through pericentric inversions play an important role in the origin of species. Certain pericentric inversions are too minute to be detected cytogenetically, thus hindering the complete reconstruction of hominoid phylogeny. The advent of the fluorescence in situ hybridization (FISH) technique has facilitated the identification of many chromosomal segments, even at the single gene level. Therefore the cosmid probe for Prader-Willi (PWS)/Angelman syndrome to the loci on human chromosome 15 [ql 1-12] is being used as a marker to highlight the complementary sequence in higher primates. We hybridized metaphase chromosomes of chimpanzee (PTR), gorilla (GGO), and orangutan (PPY) with this probe (Oncor) to characterize the chromosomal segments because the nature of these pericentric inversions remains relatively unknown. Our observations suggest that a pericentric inversion has occurred in chimpanzee chromosome (PTR 16) which corresponds to human chromosome 15 at PTR 16 band pl 112, while in gorilla (GGO 15) and orangutan (PPY 16) the bands q11-12 complemented to human chromosome 15 band q11-12. This approach has proven to be a better avenue to characterize the pericentric inversions which have apparently occurred during human evolution. Genetic divergence in the speciation process which occurs through chromosomal rearrangement needs to be reevaluated and further explored using newer techniques.Correspondence to: R.S. Verma     

Recombination between kappa chain genetic markers in the mouse

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12–1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2, 3 locus (0.3%, 95% limits, 0.05–1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41–5.4%). Lyt-2, 3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2, 3 loci as (Lyt-2, 3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the V_k-(ser) subgroup (controlled by Igk-Ef1) and V_k–1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state (NAK) and should be suitable for V _k gene mapping studies.Abbreviations C complement - C_H constant region of the Ig heavy chain - CI cytotoxicity index - DNP dinitrophenyl - FMF flow microfluorimetry - IEF isoelectric focusing - IF immunofluorescence - Ig immunoglobulin - KLH keyhole limpet hemocyanin - V_H variable region of the Ig heavy chain - V_k variable region of the Ig kappa chain - V-region variable region     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [d-Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Breeding for the Digenic Apple Resistance to Scab

The results of the apple breeding for digenic resistance to scab (1979–2000), which is more long-term than the monogenic breeding, have been reviewed. The hybrid seeds obtained from the reciprocal crossing between the V _f and V _m resistance gene donors served as the original material. The seed progeny yielded by backcrosses between these hybrids and the susceptible cultivars were examined (age—first true leaves) using inoculation in a greenhouse. The following criteria were proposed for breeding parental forms and the cultivars of the V _f v _f V _m v _m genotype: (1) phenotype segregation in the progeny of three resistant: one susceptible seedling and (2) the presence in the progeny of two types of resistant seedlings: those with the class 1 resistance (points and spots—hypersensitivity) typical of the V _m gene and those with the class 2–3 resistance (chlorotic and necrotic spots) typical of the V _f gene.     

A linkage map of the mouse immunoglobulin lambda light chain locus

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes. The order of the lambda gene segments is V2-J2C2J4C4-V1-J3C3J1C1, as was originally proposed. No evidence for nonmethylated CpG rich areas (HTF islands) within the region was found. Fine mapping using the 1, 3 rearranged cell line J558 mapped the gap between the V and J-C gene segments in the lambda 1 gene cluster (VI-J3C3JIC1) to approximately 70 kb. The similar distance (60–100 kb) found in the lambda 2 gene cluster (V2-J2C2J4C4) is further evidence that duplication of an ancestral locus occurred.     

Human genome dispersal and evolution of 4q35 duplications and interspersed LSau repeats

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.     

A rat homolog of the mouse deafness mutant jerker (je)

An autosomal recessive deafness mutant was discovered in our colony of Zucker (ZUC) rats. These mutants behave like shaker-waltzer deafness mutants, and their inner ear pathology classifies them among neuroepithelial degeneration type of deafness mutants. To determine whether this rat deafness mutation (−) defines a unique locus or one that has been previously described, we mapped its chromosomal location. F₂ progeny of (Pbrc:ZUC × BN/Crl) A/a B/b H/h+/− F₁ rats were scored for coat color and behavioral phenotypes. Segregation analysis indicated that the deafness locus might be loosely linked with B on rat Chromosome (Chr) 5 (RNO5). Therefore, 40 −/− rats were scored for BN and ZUC alleles at four additional loci, D5Mit11, D5Mit13, Oprd1, and Gnb1, known to map to RNO5 or its homolog, mouse Chr 4 (MMU4). Linkage analysis established the gene order (cM distance) as D5Mit11–(19.3)–B–(17.9)–D5Mit13–(19.2)–Oprd1–(21.5) − (1.2) Gnb1, placing the deafness locus on distal RNO5. The position of the deafness locus on RNO5 is similar to that ofjerker (je) on MMU4; the phenotypes and patterns of inheritance of the deafness mutation and je are also similar. It seems likely that the mutation affects the rat homolog of je. The rat deafness locus should, therefore, be named jerker and assigned the gene symbol Je. Received: 13 June 1995 / Accepted: 4 January 1996