Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 2--Chromosomal location of a satellite DNA

Studies about composition of repetitive sequences and their chromosomal location have been helpful to evolutionary studies in many distinct organisms. In order to keep on assessing the possible relationships among different cytotypes of Astyanax fasciatus (Teleostei, Characiformes) in the Mogi-Gua?u River (S?o Paulo State, Brazil), C-banding, chromomycin A(3) staining, and fluorescent in situ hybridization with a repetitive DNA sequence (As51) isolated from Astyanax scabripinnis were performed in the present work. The constitutive heterochromatin was distributed in terminal regions on long arms of submetacentric, subtelocentric, and acrocentric chromosomes and in the terminal region on short arms of a pair of submetacentric chromosomes in both standard cytotypes. This latter heterochromatic site was also GC-rich, as revealed by chromomycin A(3) staining, corresponding to the nucleolar organizer region (NOR), as shown by previous studies. The sites of the satellite As51 DNA were located in terminal regions on long arms of several chromosomes. Some variant karyotypic forms, which diverge from the two standard cytotypes, also presented distinctive chromosomes carrying As51 satellite DNA. It is possible that the standard 2n = 46 cytotype represents an invader population in the Mogi-Gua?u River able to interbreed with the resident standard 2n = 48 cytotype. Therefore, the variant karyotypes would be related to a possible viable offspring, where complementary chromosomal rearrangements could favor new locations of the satellite DNA analyzed.     

Chromosomal localization of the major satellite DNA family (FA-SAT) in the domestic cat

A major satellite DNA sequence was isolated from the cat genome and its sequencing data revealed homology to the FA-SAT family. In situ hybridization of the cat satellite DNA and telomeric sequences to cat chromosomes, together with staining of constitutive heterochromatin, allowed the physical mapping of the FA-SAT sequences, and also an overall constitutive heterochromatin study in cat chromosomes.     

Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking.

Transgenes can affect transgenic mice via transgene expression or via the so-called positional effect. DNA sequences can be localized in chromosomes using recently established mouse genomic databases. In this study, we describe a chromosomal mapping method that uses the genomic walking technique to analyze genomic sequences that flank transgenes, in combination with mouse genome database searches. Genomic DNA was collected from two transgenic mouse lines harboring pCAGGS-based transgenes, and adaptor-ligated, enzyme restricted genomic libraries for each mouse line were constructed. Flanking sequences were determined by sequencing amplicons obtained by PCR amplification of genomic libraries with transgene-specific and adaptor primers. The insertion positions of the transgenes were located by BLAST searches of the Ensembl genome database using the flanking sequences of the transgenes, and the transgenes of the two transgenic mouse lines were mapped onto chromosomes 11 and 3. In addition, flanking sequence information was used to construct flanking primers for a zygosity check. The zygosity (homozygous transgenic, hemizygous transgenic and non-transgenic) of animals could be identified by differential band formation in PCR analyses with the flanking primers. These methods should prove useful for genetic quality control of transgenic animals, even though the mode of transgene integration and the specificity of flanking sequences needs to be taken into account.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列

目的为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息,应用接头PCR克隆整合位点旁侧序列。方法小鼠基因组总DNA酶解后与设计的接头片段连接,根据慢病毒的LTR序列设计巢式PCR引物,克隆转基因小鼠整合位点旁侧序列。结果成功克隆到转基因小鼠整合位点的旁侧序列,经过测序定位于小鼠染色体上。结论作为反向PCR的改进,本方法可用于转基因小鼠整合位点旁侧序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity

When transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method-TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites. By way of example, using this approach, three separate integration sites were found (on chromosomes 8, 15 and 17) in one transgenic founder. As the sites on chromosomes 8 and 15 failed to segregate in any subsequent progeny, whole chromosome paints were done to determine if translocations involving chromosomes 8 and 15 occurred at the time of transgene integration. Whole chromosome painting could not detect translocations, suggesting that the rearrangements likely involve only small stretches of chromosomes. Site-specific primers were used to identify the progeny carrying only one integration site; these mice were then used as sub-founders for subsequent breedings. Integration site specific primers were used to distinguish homozygous progeny from heterozygotes. TAIL-PCR thus provides an easy and reliable way to (1) identify multiple integration sites in transgenic founders, (2) select breeders with one integration site, and (3) determine zygosity in subsequent progeny. Use of this strategy may also be considered to map integration sites in situations of unexpected phenotype or embryonic lethality while creating new transgenic mice.     

Rapid,localized amplification of a unique satellite DNA family in the rodent Microtus chrotorrhinus

A novel satellite DNA family (called MSAT-2570) was isolated and characterized from the rodent Microtus chrotorrhinus. With a length of 2,570 bp the repeat unit is among the largest yet reported in mammals and comprises a series of short direct and inverted repeats. These repeat motifs may prevent nucleosome formation or represent an endless source of genetic variation. Restriction enzyme digestion using the two pairs of isoschizomers HpaII/MspI and MboI/Sau3AI demonstrated tissue specific differences in satellite DNA methylation that may reflect variable chromatin conformation or differences in patterns of gene expression. The sex chromosomes of M. chrotorrhinus are unusually large in size among mammals, comprising 15%–20% of the karyotype and containing large blocks of heterochromatin. In situ hybridization of the satellite DNa revealed chromosomal localization predominantly to sex chromosome heterochromatin. A survey of related rodents including three congeneric species also with giant sized sex chromosomes demonstrated that MSAT-2570 is present only in the genome of M. chrotorrhinus. However, another previously reported satellite DNA also isolated from M. chrotorrhinus has been shown to reside on sex chromosome heterochromatin in one of the other three species, indicating that these giant blocks of heterochromatin are complex in structure and comprise multiple, unrelatined satellite DNA families.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

Satellite DNA and speciation: A species specific satellite DNA of Drosophila guanchel

The heterochromatin of the chromosomes of Drosophila gunche consists mainly of a satellite DNA composed of multiple, tandemly arranged copies of a 290 b p basic sequence. Five clones containing one or two copies of the basic unit were sequenced. As expected from CsCl density centrifugation and AT specific staining of mitotic chromosomes the sequence is AT rich. The average nucleotid variability between the cloned sequences is 11.6%. In situ hybridization on the mitotic chromosomes revealed, that this satellite DNA is present in the centromeric regions of all chromosomes but the Y. The nucleotide variability between copies of different tandem clusters seems to be higher than between members of the same cluster. The copy number of the sequence in the haploid genome was estimated to be approximately 80000. The sequence is species specific and is not present in the genome of sibling species D. subobscura and D. madeiren-sis. The evolutionary origin of the satellite DNA and its possible role in species formation is discussed.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.     

Karyotype and genome characterization in four cartilaginous fishes

Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.     

Association of transgene integration sites with chromosome rearrangements in hexaploid oat

Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal breakage and rearrangements. Received: 29 July 1999 / Accepted: 9 November 1999     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.     

The Drosophila Salivary Gland Chromocenter Contains Highly Polytenized Subdomains of Mitotic Heterochromatin

Peri-centromeric regions of Drosophila melanogaster chromosomes appear heterochromatic in mitotic cells and become greatly underrepresented in giant polytene chromosomes, where they aggregate into a central mass called the chromocenter. We used P elements inserted at sites dispersed throughout much of the mitotic heterochromatin to analyze the fate of 31 individual sites during polytenization. Analysis of DNA sequences flanking many of these elements revealed that middle repetitive or unique sequence DNAs frequently are interspersed with satellite DNAs in mitotic heterochromatin. All nine Y chromosome sites tested were underrepresented >20-fold on Southern blots of polytene DNA and were rarely or never detected by in situ hybridization to salivary gland chromosomes. In contrast, nine tested insertions in autosomal centromeric heterochromatin were represented fully in salivary gland DNA, despite the fact that at least six were located proximal to known blocks of satellite DNA. The inserted sequences formed diverse, site-specific morphologies in the chromocenter of salivary gland chromosomes, suggesting that domains dispersed at multiple sites in the centromeric heterochromatin of mitotic chromosomes contribute to polytene β-heterochromatin. We suggest that regions containing heterochromatic genes are organized into dispersed chromatin configurations that are important for their function in vivo.     

Organization of the 378 bp satellite repeat in terminal heterochromatin of Allium fistulosum

Telomeres, DNA-protein structures, are important elements of the eukaryotic chromosome. Telomeric regions of the majority of higher plants contain heptanucleotides TTTAGGG arranged into a tandem repeat. However, some taxa have no such repeats. These are some species of lilies (Lilium) and onions (Allium). For example, terminal regions of chromosomes of Spanish onion (Allium fistulosum) contain satellite DNA whose unit repeats are 380 bp in length, and the short arm of its chromosome 8 contains rDNA repeats. This study deals with the terminal heterochromatin and organization of the satellite repeat in A. fistulosum. Fluorescent in situ hybridization (FISH) was used to locate the satellite DNA on chromosomes and on extended DNA of A. fistulosum. Nonsatellite DNA was found in the structure of telomeric repeat. Polymerase chain reaction (PCR) and Southern hybridization were used for analysis of terminal heterochromatin. Various rearrangements were found in the satellite repeat. The roles of retrotransposones and microsatellites in the formation of terminal heterochromatin are discussed.     

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin.     

Satellite DNA probes of Alstroemeria longistaminea (Alstroemeriaceae) paint the heterochromatin and the B chromosome,reveal a G-like banding pattern,and point to a strong structural karyotype conservation

Alstroemeria species present a well-conserved and asymmetric karyotype. The genus is divided into a Chilean clade, rich in heterochromatin, and a Brazilian clade, poor in heterochromatin. We investigated the distribution of the main repetitive sequences in the chromosomes of the Brazilian species A. longistaminea (2n = 16 + 0-6B) aiming to evaluate the role played by these sequences on the structural organization of the karyotype. In situ hybridization of the three most abundant retrotransposons, corresponding to ~ 45% of the genome, was uniformly distributed. Three satellite DNA sequences, representing near half of the whole satellite fraction (1.93% of the genome), were mainly concentrated on the heterochromatin and one of them painted the whole B chromosome. Noteworthy, some satellites were located on euchromatin, either dispersed or concentrated in clusters along the chromosomes, revealing a G-band-like pattern. The two satellites that presented more C-band- and G-band-like labeling were also hybridized in situ in two other Alstroemeria species. They revealed astonishing similar patterns of distribution, indicating an unusually structural karyotype conservation among Brazilian species.

     

Organization and dynamics of satellite and telomere DNAs in Ascaris: implications for formation and programmed breakdown of compound chromosomes

In the nematode genus Ascaris the germline genome contains considerable amounts of extra DNA, which is discarded from the somatic founder blastomeres during early cleavage. In Parascaris univalens the haploid germline genome is contained in one large compound chromosome, which consists of a euchromatic region containing the somatic genome flanked by large blocks of heterochromatin. Fluorescence in situ hybridization of fractions of the germline-limited satellite DNA revealed two highly repeated sequence families establishing the entire heterochromatin (HET blocks). The repeats, a pentanucleotide, TTGCA, and a decanucleotide, TTTGTGCGTG, constitute separate segments of the HET blocks. The blocks are polymorphic in length and, hence, in copy number of the repeats, and the arrangement of the segments. The numerous sequence variants of both repeats display a disperse distribution. The type and rate of base substitutions within both repeat units depend on position. Prior to the elimination process in presomatic cells, termed chromatin diminution, the chromosomes undergo differential mitotic condensation. Interstitial 'chromatin linkers' flanking the prospective numerous somatic chromosomes remain entirely decondensed. The somatic chromosomes are released from the plurivalent chromosomes via excision of the linkers at onset of anaphase, followed by exclusion of the akinetic linker chromatin and HET blocks from the daughter nuclei. In Ascaris suum, the germline-limited satellite, which consists of one 123 bp repeat, is scattered throughout the numerous chromosomes in small heterochromatic knobs of variable sizes, residing at chromosomal ends and/or intercalary positions. The programmed breakage, which appears to proceed in a similar manner to that in P. univalens, results in the loss of all heterochromatic knobs, accompanied by an increase in chromosome number. In both species, all germline chromosomes are capped by tracts of TTAGGC repeats. In P. univalens, such telomeric tracts also occur at the termini of the euchromatic intercalary regions. Upon diminution all telomeric tracts are discarded. De novo telomere addition occurs in all somatic cell lineages of both species. The presented data shed light on the evolutionary history of chromosome aggregation and satellite DNA formation, and putative mechanisms involved in the process of site-directed breakage to reestablish stable somatic chromosomes.     

Organization of the 378-bp Satellite Repeat in Terminal Heterochromatin of Allium fistulosum

Telomeres, DNA–protein structures, are important elements of the eukaryotic chromosome. Telomeric regions of the majority of higher plants contain heptanucleotides TTTAGGG arranged into a tandem repeat. However, some taxa have no such repeats. These are some species of Liliaceae and Alliaceae. For example, terminal regions of chromosomes of bunching onion (Allium fistulosum) contain satellite DNA whose unit repeats are 380 bp in length, and the short arm of its chromosome 8 contains rDNA repeats. This study deals with the terminal heterochromatin and organization of the satellite repeat in A. fistulosum. Fluorescent in situ hybridization (FISH) was used to locate the satellite DNA on chromosomes and on extended DNA of A. fistulosum.Nonsatellite DNA was found in the structure of telomeric repeat. Polymerase chain reaction (PCR) and Southern hybridization were used for analysis of terminal heterochromatin. Various rearrangements were found in the satellite repeat. The roles of retrotransposons and microsatellites in the formation of terminal heterochromatin are discussed.