Chromosome banding in Amphibia. XXIV. The B chromosomes of Gastrotheca espeletia (Anura,Hylidae)

The mitotic chromosomes of an Ecuadorian population of the marsupial frog Gastrotheca espeletia were analyzed by means of banding techniques and fluorescence in situ hybridization. This species is characterized by unusual supernumerary (B) chromosomes. The maximum number of B chromosomes is 9 and they occur in three different morphological types. Banding analyses show that the B chromosomes are completely heterochromatic, consist of AT base pair-rich repeated DNA sequences, replicate their DNA in very late S-phase of the cell cycle, and are probably derived from a centromeric or paracentromeric region of a standard (A) chromosome. Exceptionally, the B chromosomes carry 18S + 28S ribosomal RNA genes and the conserved vertebrate telomeric DNA sequence appears to be underrepresented. Flow cytometric measurements of the nuclear DNA content differentiate between individuals with different numbers of B chromosomes. Significantly more B chromosomes are present in female than in male animals.     

B chromosome ancestry revealed by histone genes in the migratory locust

In addition to the standard set of chromosomes (A), about 15% of eukaryote genomes carry B chromosomes. In most cases, B chromosomes behave as genomic parasites being detrimental for the individuals carrying them and prospering in natural populations because of transmission advantages (drive). B chromosomes are mostly made up of repetitive DNA sequences, especially ribosomal DNA (rDNA), satellite DNA and mobile elements. In only two cases have B chromosomes been shown to carry protein-coding genes. Although some B chromosomes seem to have derived from interspecific hybridisation, the most likely source of B chromosomes is the host genome itself, but the specific A chromosome being the B ancestor has not been identified in any B-containing species. Here, we provide strong evidence for B chromosome ancestry in the migratory locust, based on the location of genes for the H3 and H4 histones in the B chromosome and a single A chromosome pair (i.e. the eighth in order of decreasing size). The high DNA sequence similarity of A and B chromosome H3–H4 genes supports B-origin from chromosome 8. The higher variation shown by B sequences, compared to A sequences, suggests that B chromosome sequences are most likely inactive and thus less subjected to purifying selection. Estimates of time of divergence for histone genes from A and B chromosomes suggest that B chromosomes are quite old (>750,000 years), showing the B-chromosome ability to persist in natural populations for long periods of time.     

The B chromosomes in Brachycome

This review presents a historical account of studies of B chromosomes in the genus Brachycome Cass. (synonym: Brachyscome) from the earliest cytological investigations carried out in the late 1960s though to the most recent molecular analyses. Molecular analyses provide insights into the origin and evolution of the B chromosomes (Bs) of Brachycome dichromosomatica, a species which has Bs of two different sizes. The larger Bs are somatically stable whereas the smaller, or micro, Bs are somatically unstable. Both B types contain clusters of ribosomal RNA genes that have been shown unequivocally to be inactive in the case of the larger Bs. The large Bs carry a family of tandem repeat sequences (Bd49) that are located mainly at the centromere. Multiple copies of sequences related to this repeat are present on the A chromosomes (As) of related species, whereas only a few copies exist in the A chromosomes of B. dichromosomatica. The micro Bs share DNA sequences with the As and the larger Bs, and they also have B-specific repeats (Bdm29 and Bdm54). In some cases repeat sequences on the micro Bs have been shown to occur as clusters on the A chromosomes in a proportion of individuals within a population. It is clear that none of these B types originated by simple excision of segments from the A chromosomes.     

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.     

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.     

Gene transfer and gene mapping in mammalian cells in culture

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.     

Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores. [Reviewing Editor: Dr. Martin Kreitman]     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research.

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

Localization of three DNA segments encompassing tRNA genes to human chromosomes 1, 5, and 16: proposed mechanism and significance of tRNA gene dispersion

The chromosomal locations of three cloned human DNA fragments encompassing tRNA genes have been determined by Southern analysis of human-rodent somatic cell hybrid DNAs with subfragments from these cloned genes and flanking sequences used as hybridization probes. These three DNA segments have been assigned to human chromosomes 1, 5, and 16, and homologous sequences are probably located on chromosome 14 and a separate locus on chromosome 1. These studies, combined with previous results, indicate that tRNA genes and pseudogenes are dispersed on at least seven different human chromosomes and suggest that these sequences will probably be found on most, if not all, human chromosomes. Short (8-12 nucleotide) direct terminal repeats flank many of the dispersed tRNA genes. The presence of these flanking repeats, combined with the dispersion of tRNA genes throughout the human genome, suggests that many of these genes may have arisen by an RNA-mediated retroposition mechanism. The possible functional significance of this gene dispersion is considered.     

A single, recent origin of the accessory B chromosome of the grasshopper Eyprepocnemis plorans

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species.     

Organization of the genome and gene expression in a nuclear environment lacking histones and nucleosomes: the amazing dinoflagellates

Dinoflagellates are fascinating protists that have attracted researchers from different fields. The free-living species are major primary producers and the cause of harmful algal blooms sometimes associated with red tides. Dinoflagellates lack histones and nucleosomes and present a unique genome and chromosome organization, being considered the only living knockouts of histones. Their plastids contain genes organized in unigenic minicircles. Basic cell structure, biochemistry and molecular phylogeny place the dinoflagellates firmly among the eukaryotes. They have G1-S-G2-M cell cycles, repetitive sequences, ribosomal genes in tandem, nuclear matrix, snRNAs, and eukaryotic cytoplasm, whereas their nuclear DNA is different, from base composition to chromosome organization. They have a high G + C content, highly methylated and rare bases such as 5-hydroxymethyluracil (HOMeU), no TATA boxes, and form distinct interphasic dinochromosomes with a liquid crystalline organization of DNA, stabilized by metal cations and structural RNA. Without histones and with a protein:DNA mass ratio (1:10) lower than prokaryotes, they need a different way of packing their huge amounts of DNA into a functional chromatin. In spite of the high interest in the dinoflagellate system in genetics, molecular and cellular biology, their analysis until now has been very restricted. We review here the main achievements in the characterization of the genome, nucleus and chromosomes in this diversified phylum. The recent discovery of a eukaryotic structural and functional differentiation in the dinochromosomes and of the organization of gene expression in them, demonstrate that in spite of the secondary loss of histones, that produce a lack of nucleosomal and supranucleosomal chromatin organization, they keep a functional nuclear organization closer to eukaryotes than to prokaryotes.     

Gene transfer and gene mapping in mammalian cells in culture

Summary The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being employed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probes. Recently, single genes have been clones into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. Supported by NIH grants HD 05196 and GM 20454 and by MOD grants 1-485 and 1-692.     

B chromosomes in Nierembergia aristata (Solanaceae): nucleolar activity and competition with the A chromosomes

B chromosomes are additional dispensable chromosomes that may be present in some individuals, populations, or species, which have probably arisen from the A chromosomes but follow their own evolutionary pathway. Supposedly, B chromosomes do not contain major genes except for ribosomal DNA (rDNA) sequences that have been mapped on the supernumerary chromosomes of many plants and animals. This paper is a new report of B chromosome occurrence in plants. B chromosomes with nucleolar organizing regions (NORs) were found in a diploid sample of Nierembergiaaristata D. Don (sub nom. N. stricta Miers) (2n = 2x = 16). This is an extreme case in which B chromosomes possess not only strong nucleolar activity, as revealed by conventional staining methods, AgNOR and fluorescence banding, and fluorescent in situ hybridization (FISH), but also show nucleolar competition with the A chromosomes. The observed phenomenon could be analogous to the nucleolar dominance or 'differential amphiplasty' phenomenon that occurs in interspecific hybrids.     

Chromosome banding in amphibia. XXIII. Giant W sex chromosomes and extremely small genomes in Eleutherodactylus euphronides and Eleutherodactylus shrevei (Anura,Leptodactylidae)

Highly differentiated, heteromorphic ZZ female symbol /ZW male symbol sex chromosomes were found in the karyotypes of the neotropical leptodactylid frogs Eleutherodactylus euphronides and E. shrevei. The W chromosomes are the largest heterochromatic, female-specific chromosomes so far discovered in the class Amphibia. The analyses of the banding patterns with AT- and GC base-pair specific fluorochromes show that the constitutive heterochromatin in the giant W chromosomes consists of various categories of repetitive DNA sequences. The W chromosomes of both species are similar in size, morphology and banding patterns, whereas their Z chromosomes exhibit conspicuous differences. In the cell nuclei of female animals, the W chromosomes form very prominent chromatin bodies (W chromatin). DNA flow cytometric measurements demonstrate clear differences in the DNA content of male and female erythrocytes caused by the giant W chromosome, and also shows that these Eleutherodactylus genomes are among the smallest of all amphibian genomes. The importance of the heteromorphic ZW sex chromosomes for the study of Z-linked genes, the similarities and differences of the two karyotypes, and the significance of the exceptionally small genomes are discussed.     

The Giardia lamblia genome

Giardia lamblia is a protozoan parasite of humans and other mammals that is thought to be one of the most primitive extant eukaryotic organisms. Although distinctly eukaryotic, it is notable for its lack of mitochondria, nucleoli, and perixosomes. It has been suggested that Giardia spp. are pre-mitochondriate organisms, but the identification of genes in G. lamblia thought to be of mitochondrial origin has generated controversy regarding that designation. Giardi lamblia trophozoites have two nuclei that are identical in all ways that have been studied. They are polyploid with at least four, and perhaps eight or more, copies of each of five chromosomes per organism and have an estimated genome complexity of 1.2x10(7)bp of DNA, and GC content of 46%. There is evidence for recombination at the telomeres of some of the chromosomes, and multiple size variants of single chromosomes have been identified within cloned isolates. However, the internal regions of the chromosomes demonstrate no evidence of recombination. For example, there is no evidence for control of vsp gene expression by DNA recombination, and no evidence for rapid mutation in the vsp genes. Single pass sequences of approximately 9% of the G. lamblia genome have already been obtained. An ongoing genome project plans to obtain approximately 95% of the genome by a random approach, as well as a complete physical map using a bacterial artificial chromosome library. The results will facilitate a better understanding of the biology of Giardia spp. as well as their phylogenetic relationship to other primitive organisms.