Stable methylation patterns in interspecific antelope hybrids and the characterization and localization of a satellite fraction in the Alcelaphini and Hippotragini

Conflicting data has recently appeared concerning altered methylation patterns in interspecific mammalian hybrids and the potential this may hold for driving karyotypic evolution. We report no detectable methylation difference in the genomic DNA of different interspecific F1 antelope hybrids (family Bovidae) and their parent species using the methylation-sensitive enzyme HpaII and its methylation insensitive isoschizomer MspI. However, both enzymes released a tandemly repeated satellite array. Characterization of the repeat using Southern blotting and a combination of sequencing, fluorescence in-situ hybridization (FISH) and C-banding, shows some similarity in the family of repeats between the hybridizing antelope species groups, and that the satellite is localized in the centromeric C-band positive regions of the chromosomes. Moreover, although there is little meaningful sequence homology with the well characterized bovine 1.715 satellite DNA, there is 86% sequence similarity with the sheep/goat satellite I, suggesting that they are related and are likely to have originated and evolved separately from the bovine unit.     

Interstitial colocalization of two cervid satellite DNAs involved in the genesis of the Indian muntjac karyotype

A number of repetitive DNA clones were generated from PCR amplifications of Indian muntjac genomic DNA using primer sequences derived from a white tailed deer satellite II DNA sequence. One clone (Mmv-0.7) was characterized and shown to be a cervid satellite II DNA clone. Multiple colored FISH studies with cervid satellite I (C5) and this satellite II clone (Mmv-0.7) to Chinese muntjac metaphase chromosomes localized both satellite DNAs at the pericentromeric regions of all chromosomes except for chromosome 3 and the Y chromosome, whereas chromosome 3 exhibited pericentromeric satellite II DNA only. Where distinguishable, the pericentromeric satellite II signals appeared terminally oriented with respect to satellite I. Six pairs of Chinese muntjac autosomes had interstitial satellite I sites with four of these autosomal pairs (chromosomes 1, 2 and two other smaller autosomal pairs) also exhibiting interstitial satellite II signals. An interstitial site on the X chromosome was found to have satellite II signals. For the Indian muntjac chromosomes, FISH studies revealed a pericentromeric hybridization for satellites I and II as well as 27 distinct interstitial hybridization sites, each having at least one of the satellite DNAs. These data were used to more precisely define the chromosome fusion-associated breakpoints that presumably led to the formation of the present-day Indian muntjac karyotype. It further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a 2n=70 ancestral karyotype rather than from an intermediate 2n=46 Chinese muntjac-like karyotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization and chromosomal distribution of novel satellite DNA sequences of the lesser rhea (Pterocnemia pennata) and the greater rhea (Rhea americana)

Two different types of novel satellite DNA (stDNA) sequences were cloned from the lesser rhea (Ptercnemia pennata) and the greater rhea (Rhea americana) after digestion of genomic DNAs with a restriction endonuclease Pvu II, and characterized by filter hybridization and in-situ hybridization to metaphase chromosomes. These nucleotide sequences consisted of GC-rich 288-bp and 332-bp repeated elements in P. pennata and 288-bp and 336-bp repeated elements in R. americana, all of which were organized in tandem arrays in the genome. The 288-bp and 332-bp elements of P. pennata displayed strong sequence similarity with the 288-bp and 336-bp elements of R. americana, respectively. The 332-bp and 336-bp elements were located on almost all the microchromosomes in both the species. The other type of repeated elements, the 288-bp element, was located on four and nine pairs of microchromosomes in P. pennata and R. americana, respectively. All the stDNA sequences were not crosshybridized to genomic DNAs of another three ratite species, ostrich (Struthio camelus), cassowary (Casuarius casuarius) and emu (Dromaius novaehollandiae), suggesting that these stDNA sequences are conserved in the same family but fairly divergent among the different families of Struthioniformes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diversity of DNA beta,a satellite molecule associated with some monopartite begomoviruses

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.     

Detection of human chromosomal abnormalities using a new technique combining 4',6-diamidino-2-phenyl-indole staining and image analysis

Chromosomal abnormalities are associated with a variety of diseases. We have developed a new technique for detecting chromosomal abnormalities, and the technique combines conventional 4',6-diamidino-2-phenyl-indole staining (DAPI) with image analysis. The image analysis consists of two simple steps: deconvolution and three-dimensional reconstruction. The technique has been reported for analyzing plant chromosomes but has not been applied to analyze human chromosomes yet. To test the technique, we analyzed five translocations: 46,XX,t(3;21)(12;18), 46,XX,t(11;22), 46,XY,t(7;22), 46,XY,t(11;18), and 46,XY,t(3;7). The results showed that the karyotype of the 46,XX,t(3;21)(12;18) was 46,XX,t(3;21)(q11.1;p13),t(12;18) (q21.2;q23), and the karyotypes of the 46,XX,t(11;22), 46,XY,t(7;22), 46,XY,t(11;18), and 46,XY,t(3;7) were 46,XX,t(11;22)(q23;q12.1); 46,XY,t(7;22)(q32;q13.2); 46,XY,t(11;18)(q13.3;q23), and 46,XY,t(3;7)(q22.1;p13), respectively. The identity of derivative chromosomes involved in the translocations was verified by chromosome painting as well as FISH analyses with centromere probes. The new technique has two advantages: the procedure is simple and convenient, and the results are accurate. The technique has the potential to be used in cytogenetic studies and clinical diagnosis of human diseases in the future.     

Organization and molecular cytogenetics of a satellite DNA family fromHoplias malabaricus (Pisces,Erythrinidae)

The chromosomes of the primitive South American teleost fishHoplias malabaricus have been analyzed by classical cytogenetic (C-, AgNOR-, Hoechst 33258-, and Q-banding) techniques. A highly repetitive DNA family has been cloned and sequenced. It is a tandemly repeated sequence of about 355 bp, yielding an overall base pair composition of 67% AT with long runs of >50% As and 70% Ts. Analysis of sequence variation has allowed the further categorization ofHoplias satellite DNA into two evolutionarily related subfamilies A and B, distinguishable by characteristic insertions and deletions within this 355-bp monomer. Subfamily A satellite is found (in diverged form) at the centromeres of mostH. malabaricus chromosomes. Sequence variants are clustered on specific chromosomal subsets. Subfamily B satellite is highly specific for the paracentromeric heterochromatin on one particular chromosome pair by fluorescencein situ hybridization. These results indicate that theHoplias satellite DNA family has evolved in a concerted manner predominantly via recombination events involving homologous, rather than non-homologous chromosome regions. The clones isolated here may be useful for the molecular, genetic, and cytological analysis of the genusHoplias.     

CENP-B box and pJα sequence distribution in human alpha satellite higher-order repeats (HOR)

Using our Key String Algorithm (KSA) to analyze Build 35.1 assembly we determined consensus alpha satellite higher-order repeats (HOR) and consensus distributions of CENP-B box and pJα motif in human chromosomes 1, 4, 5, 7, 8, 10, 11, 17, 19, and X. We determined new suprachromosomal family (SF) assignments: SF5 for 13mer (2211 bp), SF5 for 13mer (2214 bp), SF2 for 11mer (1869 bp), SF1 for 18mer (3058 bp), SF3 for 12mer (2047 bp), SF3 for 14mer (2379 bp), and SF5 for 17mer (2896 bp) in chromosomes 4, 5, 8, 10, 11, 17, and 19, respectively. In chromosome 5 we identified SF5 13mer without any CENP-B box and pJα motif, highly homologous (96%) to 13mer in chromosome 19. Additionally, in chromosome 19 we identified new SF5 17mer with one CENP-B box and pJα motif, aligned to 13mer by deleting four monomers. In chromosome 11 we identified SF3 12mer, homologous to 12mer in chromosome X. In chromosome 10 we identified new SF1 18mer with eight CENP-B boxes in every other monomer (except one). In chromosome 4 we identified new SF5 13mer with CENP-B box in three consecutive monomers. We found four exceptions to the rule that CENP-B box belongs to type B and pJα motif to type A monomers. Electronic Supplementary Material Supplementary material is available for this article at     

Proliferation conditions for human satellite cells. The fractional content of satellite cells

Primary satellite cell cultures have become an important tool as a model system for skeletal muscles. A common problem in human satellite cell culturing is fibroblast overgrowth. We combined N-CAM (Leu19) immunocytochemical staining of satellite cells (Sc) with stereological methods to estimate the fraction of Sc in culture. Evaluation of different culture conditions allowed us to find proliferation conditions preferentially for Sc: a) Sc should be cultured on surfaces coated with ECM-gel. b) Primary cell culture should be inoculated in DMEM supplemented with 10% fetal calf serum to increase cell adherence. c) Change of media to DMEM supplemented with 2% Ultroser-G and 2% FCS after 24 h.d) Before subcultivation, cells should be preplated for 30 min. The fractional content of Sc in passage four when applying this method of cultivation was 0.82 +/- 0.07 (mean +/- SE, N = 10). Our method enabled us to establish culture conditions which resulted in high Sc content despite several subcultivations. Estimation of the fractional cell content could be a useful tool for optimizing not only Sc-culturing but all cultures initially containing more cell types.     

Immunolocalization of alphaV,alpha3 and beta1 integrins in the human placenta with pre-eclampsia

Signs of pre-eclampsia are considered to be caused by maternal endothelial dysfunction due to circulating factors of placental origin. Integrins are a large family of cell surface, proteins that serve as receptors involved in cell-cell and cell-matrix interactions during placentation. Therefore, low expression of integrins or the lack of it may be encountered during pre-eclampsia. In the present study, we investigated the immunolocalisation of integrins alphaV, alpha3 and beta1 in placentas of normal and pre-eclamptic women. Thirty-two placentas from pre-eclamptic (n = 14) and normotensive (n = 18) women were used. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue specimens, using anti-alphaV, anti-alpha3 and anti-beta1 antibodies and the indirect immunoperoxidase technique. A semi-quantitative grading system (HSCORE) was used to compare immunohistochemical staining intensities. Distribution patterns of alphaV, alpha3 and beta1 integrins were detected in cytotrophoblasts and Hofbauer cells in normal and pre-eclamptic placentas. Immunostaining of alphaV and beta1 integrins was slightly decreased in pre-eclamptic samples but alpha3 integrin immunostaining was similar in pre-eclamptic and normal placentas. Decreased immunostaining of integrins in the cytotrophoblasts may considered to be a structural basis for decreased placental perfusion in pre-eclampsia.     

Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs

Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol. wt proteins. Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1 alpha and IL-1 beta exist as single genomic copies. In addition, bovine IL-1 beta mRNA is approx. 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.     

Higher-order organization and compartmentalization of satellite DNA PIM357 in species of the coleopteran genus Pimelia

The PIM357 satellite DNA family is present in 26 Pimelia taxa (Tenebrionidae, Coleoptera) with endemic congeneric species from the Canary Islands showing higher interrepeat variability than continental ones. In this paper, we compare the repetitive DNA sequences of a Canarian species that has distinct subfamilies of repeat units, P. radula ascendens, with another without such subfamilies, P. sparsa sparsa. The chromosomal localization of the repeat units and the comparison of the variability of randomly cloned monomers to the one estimated by comparing repeat units from dimers and trimers suggest the absence of satellite subfamilies in P. sparsa sparsa. Hence, the repeat units of this species seem to be uniformly and randomly distributed throughout all chromosomes out of one chromosomal pair. On the contrary, P. radula ascendens shows four divergent subfamilies of repeat units supported by several diagnostic nucleotide substitutions. These subfamilies seem to form four distinct repeat units: monomer subfamily 1, monomer subfamily 4 and two higher-order units (dimer linking subfamily 1 and 4, and dimer linking subfamily 2 and 3). Moreover, monomers of subfamily 1 are present in three chromosomal pairs only. We discuss the effect of different potential factors acting in the concerted evolution and the genomic organization of stDNA sequences in these taxa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Further data on the occurrence and evolution of satellite DNA families in the lacertid genome

This paper reports the isolation and characterization of twoHindIII repetitive DNA families from the genome of two lacertid lizards,Podarcis sicula andLacerta saxicola. These satellites did not appear to be related to each other. The consensus sequences of their monomeric units did not show any similarity, though both DNAs were A-T rich. Moreover, each of them was found only in closely related species. The monomeric unit of theHindIII DNA family isolated fromP. sicula (pLHS) showed a close resemblance to pLCS, a centromeric satellite DNA previously isolated from the same species; it was, however, mainly localized at pericentromeric, interstitial and telomeric levels. The results also provide interesting information on the systematics of the lacertids studied.     

5-杂氮胞苷和甲基硝基亚硝胍处理细胞DNA中5-甲基胞嘧啶含量的HPLC分析

应用高效液相色谱技术对5-azaCR及MNNG处理的FL、Wish及Vero-E6细胞的DNA中5—甲基胞嘧啶(~mC)含量进行直接测量。结果表明5—aza CR(2×10~(-6)mol/L)处理细胞DNA的~mC含量较对照细胞为低(P<0.01),但在MNNG处理的FL细胞(5×10~(-6)及1×10~(-5)mol/LMNNG)及Wish和Vero-E6细胞(2及3.3×10~(-5)mol/L MNNG)中,DNA的~mC含量与对照并无差异(P>0.05)。这与用HpaⅡ限制性片段长度放射活性分析法检测新复制DNA甲基化状态的结果一致。认为文献中关于细胞DNA低甲基化是化学致癌启动过程普遍规律的结论是由于实验设计的内在缺陷所致。     

Condensation behaviour of the human X chromosome in male germ cells and Sertoli cells examined by fluorescencein situ hybridization

The chromatin condensation behaviour of the human X chromosome has been studied by fluorescencein situ hybridization (FISH) analysis in germ cells and Sertoli cells of the adult testis, and comparisons are made with previous findings for the human Y chromosome and for chromosome 7. In meiotic prophase, the X chromosome can be seen to extend greatly at zygotene and to contract through pachytene into the sex vesicle. Such extension, which has also been noted for the human Y chromosome at this stage of meiosis, could be a prerequisite for XY pairing and crossing-over. Byin situ hybridization analysis, the sex chromosomes of patients with Sertoli-cell-only syndrome appear extremely contracted compared with the normally extended state of those in adult Sertoli cells of fertile men. By contrast, the state of expansion for chromosome 7 in Sertoli cells appears identical for sterile and fertile testes. This could suggest an association between gene-controlled germ cell losses and failure of expansion of the sex chromosome axes. The variable patterns of extension and contraction for the X and Y chromosome axes in germ cells and Sertoli cells might provide underlying clues to patterns of expression noted for sex-linked genes in the human testis.     

Intrachromosomal distribution of telomeric repeats in Eumops glaucinus and Eumops perotis (Molossidae, Chiroptera)

The location of chromosomal telomeric repeats (TTAGGG)_n was investigated in two species of the Molossidae family, Eumops glaucinus and Eumops perotis. The diploid chromosome number (2n) is 40 in E. glaucinus and 48 in E. perotis and the fundamental numbers (FN) are 64 and 58, respectively. It has been suggested that the E. glaucinus karyotype has evolved from the E. perotis karyotype through Robertsonian fusion events. In the present study, the telomeric sequences were detected at the termini of chromosomes in both species. In addition, E. glaucinus also displayed telomeric repeats in centromeric and pericentromeric regions in almost all biarmed chromosomes. Conversely, in E. perotis pericentromeric signals were only observed in two biarmed chromosomes. In both E. glaucinus and E. perotis, such telomeric sequences were observed as part of the heterochromatin. The interstitial sites of telomeric sequences suggest that they are remnants of telomeres of ancestral chromosomes that participated in the fusion event.     

人肝细胞癌中整合素α5,β1亚基和纤维连接蛋白mRNAs的表达

目的研究肝细胞癌（ＨＣＣ）中整合素α５、β１亚基和纤维连接蛋白（ＦＮ）ｍＲＮＡｓ表达的生物学意义。方法应用Ｎｏｒｔｈｅｒｎ印迹杂交和核酸原位杂交技术，对１５例ＨＣＣ癌组织、５例癌旁肝硬化和３例正常肝组织中整合素α５、β１亚基和ＦＮｍＲＮＡｓ表达作杂交分析。结果高分化ＨＣＣ癌组织中三种ｍＲＮＡｓ表达水平与癌旁及正常肝组织相近，而低、中分化ＨＣＣ癌组织内明显减少，甚至消失（分别Ｐ＜００１）；整合素α５亚基和ＦＮｍＲＮＡｓ主要见于癌细胞胞浆内；ＨＣＣ伴肝内浸润和／或转移组癌组织内三种ｍＲＮＡｓ水平较无浸润转移组显著下降（Ｐ＜００５）；５例伴肝内转移的ＨＣＣ癌组织中ＦＮｍＲＮＡ呈异质性表达。结论ＨＣＣ中整合素α５、β１和ＦＮ蛋白的表达变化系癌细胞基因转录水平下调的结果，可能与ＨＣＣ细胞分化、浸润和转移相关；异质性ＦＮｍＲＮＡ及其编码的ＦＮ蛋白可能在ＨＣＣ转移中有意义。     

Molecular cytogenetic analysis of heterochromatin in the chromosomes of tilapia, Oreochromis niloticus (Teleostei: Cichlidae)

The structure of the heterochromatic bands in mitotic chromosomes of the important tropical aquaculture species of tilapia, Oreochromis niloticus, was investigated by the combination of the C-banding technique, chromosomal digestion with two restriction endonucleases and fluorescence in situ hybridization (FISH) of two satellite DNAs (SATA and SATB). The tilapia chromosomes presented heterochromatic bands in the centromeres and in the short arms of almost all chromosomes that were differentially digested by the restriction endonucleases HaeIII and EcoRI. FISH of SATA showed that this satellite sequence is distributed in the centromeric region of all chromosomes of tilapia. FISH also revealed an intense hybridization signal for SATB in only one chromosome pair, but less intense signals were also present in several other pairs. The digestion of tilapia chromosomes by HaeIII and EcoRI was positively correlated with the position of SATA and SATB in chromosomes as revealed by FISH. The results obtained may be useful in future molecular and genetic studies of tilapias.     

Association of pKi-67 with satellite DNA of the human genome in early G1 cells

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.     

Ameloblastoma and adenomatoid odontogenic tumor: the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in local invasiveness and architectural characteristics

Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and a tendency toward recurrence, whereas adenomatoid odontogenic tumor (AOT) is an indolent neoplasm. The objective of the present study was to immunohistochemically analyze the role of alpha2beta1, alpha3beta1, and alpha5beta1 integrins in the cellular events and cell-matrix interactions that occur in these tumors and their consequent repercussions on the architectural arrangement and biologic behavior of these lesions. Paraffin-embedded specimens from 30 ameloblastomas (20 solid and 10 unicystic tumors) and 12 AOTs were submitted to immunohistochemistry using the catalyzed signal amplification system. A difference in the pattern of integrin expression was observed between the various histologic types of ameloblastoma. No significant difference in labeling intensity was observed between unicystic and solid ameloblastomas, but comparison between ameloblastomas and AOT showed a significantly stronger expression of alpha5beta1 integrin in the former (P < .05). Our findings suggest an important role of the integrins studied in the architectural characteristics of ameloblastomas and AOTs and a possible participation of alpha5beta1 integrin in the mechanism of local invasion of ameloblastomas.