Chromosomal rearrangement in pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops, using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.     

FISH Mapping and Identification of Zhikong Scallop (Chlamys farreri) Chromosomes

Chromosome identification is the first step in genomic research of a species, but it remains a challenge in scallops. In the present study, fluorescence in situ hybridization (FISH) mapping of 19 fosmid clones was attempted and used for chromosome identification in Zhikong scallop (Chlamys farreri Jones et Preston, 1904). Data showed that 10 clones were successfully mapped, including 7 without and 3 with C ₀ t-1 DNA. Among them, 2 represented multiple signals and made no contribution to chromosome identification. Karyotypic analysis and cohybridization indicated that the remaining 8 clones realized the identification of 8 chromosomes. All 10 clones were sequenced at both ends, which could be developed as sequence-tagged sites and used for the unification of the cytological and genetic linkage maps. This study shows that fosmid clones can benefit chromosome identification and will undoubtedly be useful for cytogenetic research in Zhikong scallop.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

The Gene Encoding the Catalytic Subunit Cα of cAMP-Dependent Protein Kinase (Locus PRKACA) Localizes to Human Chromosome Region 19p13.1

We have determined the chromosomal localization of the gene for the catalytic subunit Cα of cAMP-dependent protein kinase (locus PRKACA) to human chromosome 19 using polymerase chain reaction (PCR) and Southern blot analysis of two different somatic cell hybrid mapping panels. In addition, PCR analysis of a chromosome 19 mapping panel revealed the presence of a human Cα-specific amplification product only in cell lines containing the region 19p13.1 to 19q12. Finally, two-color fluorescencein situhybridization to metaphase chromosomes using the human Cα cDNA and human chromosome 19 inter-Alu-PCR product as probes localized the human Cα gene to chromosome region 19p13.1.     

The Mitochondrial Genomes of Two Scallops, Argopecten irradians and Chlamys farreri (Mollusca: Bivalvia): The Most Highly Rearranged Gene Order in the Family Pectinidae

Molluscs in general and bivalves in particular, exhibit an extraordinary degree of mitochondrial gene order variation when compared with other metazoans. Here, we determined the mitochondrial genomes of two scallops Argopecten irradians and Chlamys farreri. The complete mitochondrial genome of A. irradians is 16,211 nts in length and the nearly complete mitochondrial genome of C. farreri is 20,789 nts in length. Both of the genomes contain 35 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 21 transfer RNAs. In contrast to the typical animal mitochondrial genome, both of them lack one protein-coding gene atp8 and two trnSs, but show an additional copy of trnF in A. irradians and of trnM in C. farreri, respectively. Gene order and genome content were compared among the four sequenced scallops. Gene arrangement of C. farreri closely resembles that of Mizuhopecten yessoensis. However, two genomes of C. farreri and A. irradians show only three small identical gene blocks and two genomes of A. irradians and Placopecten magellanicus share only one gene block. Comparison of the gene arrangement demonstrated that the four scallops share few identical gene blocks although they belong to the same family. This feature is seldom observed in Metazoa, even in other molluscan classes. The dramatic gene rearrangement often occurs in bivalves, especially in marine bivalves. In addition, comparisons of genomic character among bivalves are also presented.     

Genetic characterization and molecular mapping of Hessian fly resistance genes derived from Aegilops tauschii in synthetic wheat

Two synthetic hexaploid wheat lines (×Aegilotriticum spp., 2n = 6x = 42, genomes AABBDD), SW8 and SW34, developed from the crosses of the durum wheat cultivar Langdon (Triticum turgidum L. var. durum, 2n = 4x = 28, genomes AABB) with two Aegilops tauschii Cosson accessions (2n = 2x = 14, genome DD), were determined to carry Hessian fly [Mayetiola destructor (Say)] resistance genes derived from the Ae. tauschii parents. SW8 was resistant to the Hessian fly biotype Great Plains (GP) and strain vH13 (virulent to H13). SW34 was resistant to biotype GP, but susceptible to strain vH13. Allelism tests indicated that resistance genes in SW8 and SW34 may be allelic to H26 and H13 or correspond to paralogs at both loci, respectively. H26 and H13 were localized to chromosome 4D and 6D, respectively, in previous studies. Molecular mapping in the present study, however, assigned the H26 locus to chromosome 3D rather than 4D. On the other hand, mapping of the resistance gene in SW34 verified the previous assignment of the H13 locus to chromosome 6D. Linkage analysis and physical mapping positioned the H26 locus to the chromosomal deletion bin 3DL3-0.81–1.00. A linkage map for each of these two resistance genes was constructed using simple sequence repeat (SSR) and target region amplification polymorphism (TRAP) markers.     

CHROMOSOME NUMBERS IN BROMELIACEAE

Eighty-three chromosome counts are reported for 72 taxa of the Bromeliaceae. Fifty-eight of these counts are the first known chromosome number reports for their respective taxa. A model of chromosomal evolution in the Bromeliaceae (n = 25) is presented. The model is parsimonious and consistent with existing data on meiotic chromosome numbers within the family and in the closely related Velloziaceae (n = 9). Two hypothesized paleodiploids (n = 8 and n = 9) hybridized to form a tetraploid that in turn hybridized with the n = 8 lineage. The resultant n = 25 is the extant base number for the family. Two alternative hypotheses could explain the unique extant base number (n = 17) for Cryptanthus: 1) Cryptanthus represents the paleotetraploid level, i.e., prior to the second round of hybridization, or 2) the lower number represents the result of a more recent series of aneuploid reductions from n = 25. Given the existence of intergeneric hybrids involving Cryptanthus, aneuploid reduction is the more likely interpretation.     

Histone gene transposition in the phylogeny of the Drosophila obscura group

The chromosomal location of the histone genes was determined in seven species of the Drosophila obscura group by in situ hybridization. Histone genes occur on more than one site per genome and on non-homologous chromosome elements. In addition, the metaphase karyotypes and the banding pattern of the polytene chromosomes were compared. Based on chromosomal characters, the cladogenesis of the D. obscura group was established. From the distribution of histone sites in different species, analysed in this paper and in previous studies, the phylogenetic history of histone gene transposition was derived. The molecular mechanisms responsible for the generation of new histone sites are discussed.     

Localization ofHuC(ELAVL3) to Chromosome 19p13.2 by Fluorescencein SituHybridization Utilizing a Novel Tyramide Labeling Technique

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, andHuC(HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescencein situhybridization (T-FISH) technique, we localizedHuCto chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that ofHuR(HGMW-approved symbol ELAVL1), anotherelavfamily member. Dual T-FISH analysis withHuCandHuRprobes, however, indicated distinct loci, withHuCbeing centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.     

Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis> (Bivalvia: Pectinidae)

The complete mitochondrial genome of Zhikong scallop Chlamys farreri is 21,695 bp in length and contains 12 protein-coding genes (the atp8 gene is absent, as in most bivalves), 2 ribosomal RNA genes, and 22 transfer RNA genes. The heavy strand has an overall A+T content of 58.7%. GC and AT skews for the mt genome of C. farreri are 0.337 and ?0.184, respectively, indicating the nucleotide bias against C and A. The mitochondrial gene order of C. farreri differs drastically from the scallops Argopecten irradians, Mimachlamys nobilis and Placopecten magellanicus, which belong to the same family Pectinidae. 6623 bp non-coding nucleotides exist intergenically in the mitogenome of C. farreri, with a large continuous sequence (4763 bp) between tRNA ^Val and tRNA ^Asn . Two repeat families are found in the large continuous sequence, which seems to be a common feature of scallops. Phylogenetic analysis based on 12 concatenated amino acid sequences of protein-coding genes supports the monophyly of Pectinidae and paraphyletic Pteriomorphia with respect to Heteroconchia.     

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA) n , (TTAGGG) n and ribosomal genes by one-color and two-color FISH

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA) _n , and (TTAGGG) _n , and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG) _n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA) _n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.     

Replication Timing Properties within the Mouse Distal Chromosome 7 Imprinting Cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57^Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57^Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.     

The Human Intercellular Adhesion Molecule 3 (ICAM3) Gene Is Located in the 19p13.2-p13.3 Region, Close to the ICAM1 Gene

The chromosomal location of the intercellular adhesion molecule 3 (ICAM3) gene, coding for a lymphocyte function-associated antigen (LFA)-1 counterreceptor and selectively expressed by human leukocytes, was analyzed by in situ hybridization with the cDNA coding sequence as a probe. This sequence mapped to the p13.2-p13.3 region of chromosome 19, close to the ICAM1 gene chromosomal location.     

Effects of temperature stress on NO-synthase and tyrosine hydroxylase activities in the central nervous system of bivalve molluscs

Effects of temperature stress on activities of NO-synthase (NOS) and tyrosine hydroxylase (TH) in the CNS of two species of bivalve molluscs, Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and immunocytochemistry. General and specific peculiarities in distribution and relative proportion of TH- and NO-containing neurons in the CNS nerve ganglia were revealed in norm and under stress at 30°C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been found to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In intact Chlamys farreri nipponensis, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.     

Karyotype analysis of the Russian wheat aphid, <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO₃, CMA₃, and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the “arthropod” telomeric sequence (TTAGG) _n using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO₃-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG) _n telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.     

Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.     

Detection of a Single-Locus Gene on Channel Catfish Chromosomes by In-Situ Polymerase Chain Reaction

An in-situ polymerase chain reaction (ISPCR) procedure was applied to chromosomal localization of the gene, Ig H, encoding the immunoglobulin heavy chain of channel catfish (Ictalurus punctatus). Metaphase chromosomes were prepared by a replication banding procedure and subjected to ISPCR using biotin-labeled primers. The hybridization signals were detected with an avidin-fluorescein isothiocyanate (FITC)-based method, and chromosome bands revealed by simultaneous or sequential treatment methods. Standard fluorescent in-situ hybridization (FISH) was performed on chromosome preparations to compare with the ISPCR procedure. The Ig H gene was detected at the telomeric position of a chromosome with a relative length of 3.2 ± 0.2%. The Ig H-bearing chromosome detected by the FISH method was identical to that found by ISPCR procedure. Visibility of chromosome bands was reduced by heat and salt treatments and could not be analyzed after thermocycling. Therefore, specific identity of the chromosome bearing the Ig H gene remains unknown. Banding of fish chromosomes is difficult and poses a barrier for applying current molecular techniques to physical mapping of teleost genomes. Application of the ISPCR to chromosomal mapping is new for fish species and is only in initial stages of development for higher vertebrates.     

Testis-Specific Expression of a Novel Human H3 Histone Gene

We have investigated the expression of a recently described, solitary human H3 histone gene. Using RNase protection assays, the corresponding mRNA could only be detected in RNA preparations from human testis, whereas several human cell lines and somatic tissues did not exhibit expression of this gene.In situhybridization of sections from human testis revealed expression to be confined to primary spermatocytes. In addition to H1t, this novel H3 gene, which is located on chromosome 1, is the second tissue-specific human histone gene that has been found to be expressed solely in the testis.