人端粒酶RNA的cDNA探针制备及其对胃粘膜细胞端粒酶RNA表达的检测

目的 建立人端粒酶RNA表达的检测方法。方法 制备人端粒酶RNA，(human telomeraseRNA，hTR)的cDNA探针，分别应用RNA斑点杂交与端粒重复序列扩增法(TRAP)分析检测不同胃粘膜的端粒酶RNA的表达与端粒酶活性。结果 人端粒酶RNA的cDNA探针制备成功。18例活检胃癌组织及45例手术胃癌组织RNA斑点杂交检测的阳性率均为l00％，相应TRAP分析的阳性率分别为88．89％、86．67％，低于RNA斑点杂交(P＜0．05)。同时RNA斑点杂交结果提示在非癌胃组织中随着肠化程度增高人端粒酶RNA表达也增强。结论 RNA斑点杂交检测人端粒酶RNA，具有高度的敏感性和特异性，弥补了TRAP分析敏感性不足的缺点。     

Mapping the Distribution of the Telomeric Sequence (T2AG3) n in the 2n = 14 Ancestral Marsupial Complement and in the Macropodines (Marsupialia: Macropodidae) by fluorescence in situ hybridization

In this study we test the theory that the presence of the conserved vertebrate telomeric sequence (T(2)AG(3))(n) at the centromeres of Australian marsupial 2n = 14 complements is evidence that these karyotypes are recently derived, which is contrary to the generally held view that the 2n = 14 karyotype is ancestral for Australasian and American marsupials. Here we compare the distribution of the (T(2)AG(3))( n ) sequence and constitutive heterochromatin in the presumed ancestral 2n = 14 complement and in complements with known rearrangements. We found that where there were moderate to large amounts of constitutive heterochromatin, the distribution of the (T(2)AG(3))(n) sequence reflected its presence as a native component of satellite DNA rather than its involvement in past rearrangements. The presence of centromeric heterochromatin in all Australian 2n = 14 complements therefore suggests that centromeric sites of the (T(2)AG(3))(n) sequence do not represent evidence for recent rearrangements.     

Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes,the genera <Emphasis Type="Italic">Hylobates</Emphasis> and <Emphasis Type="Italic">Symphalangus</Emphasis>

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.     

Telomeric and interstitial telomeric sequences in holokinetic chromosomes of Lepidoptera: Telomeric DNA mediates association between postpachytene bivalents in achiasmatic meiosis of females

Telomeres, besides their main role in the protection and maintenance of chromosome ends, have several other vital functions in the cell cycle. We studied their role in the achiasmatic meiosis of female Lepidoptera, insects with holokinetic chromosomes. By fluorescence in-situ hybridization (FISH) with the insect telomeric probe, (TTAGG) _n , we mapped the distribution of telomeric and interstitial telomeric sequences (ITS) in female meiotic chromosomes of two species, Orgyia antiqua with a reduced chromosome number (2n=28) and Ephestia kuehniella mutants, possessing a radiation-induced chromosome fusion in the genome (2n=59). In addition to the strong typical telomeric signals, O. antiqua displayed weaker hybridization signals in interstitial sites of pachytene bivalents. The observed ITS most probably reflect remnants of chromosomal rearrangements and support the hypothesis that the Orgyia karyotype had arisen by multiple fusions of ancestral chromosomes. On the other hand, the absence of ITS in the chromosome fusion of Ephestia indicated the loss of telomeres before the two original chromosomes fused. When the telomeric probe was amplified by enzymatic reaction with tyramid, the number of ITS observed increased in Orgyia, and a few ITS were also observed in several chromosomes of Ephestia but not in the fused chromosome. This suggests that the genomes of both species also contain ITS other than those originating from chromosome fusions. The analysis of female meiotic prophase I revealed non-homologous associations of postpachytene bivalents mediated by telomeric DNA, which were not observed in the pachytene stage. Surprisingly, in early postpachytene nuclei the telomeric associations also involved ITS, whereas later postpachytene nuclei displayed chains of bivalents interconnected only by true telomeres. This finding favours a hypothesis that telomeric associations between bivalents play a role in chromosome segregation in the achiasmatic meiosis of female Lepidoptera. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nature of telomere dimers and chromosome looping in human spermatozoa

Specific and well-organized chromosome architecture in human sperm cells is supported by the prominent interactions between centromeres and between telomeres. The telomere-telomere interactions result in telomere dimers that are positioned at the nuclear periphery. It is unknown whether composition of sperm telomere dimers is random or specific. We now report that telomere dimers result from specific interactions between the two ends of each chromosome. FISH using pairs of subtelomeric DNA probes that correspond to the small and long arms of seven human chromosomes demonstrates that subtelomeres of one chromosome are brought together. Statistical analysis confirmed that telomere associations could not result from the random proximity of DNA sequences. Therefore, chromosomes in human sperm nuclei adopt a looped conformation. This higher-order chromosome structure is most likely required for chromosome withdrawal/decondensation during the early fertilization events leading to zygote formation. These individuals contributed equally to the work     

Telomere structure, function and maintenance in Arabidopsis

The stability of eukaryotic genomes is provided in part by the integrity of telomeres, the nucleoprotein caps on the ends of chromosome. Recent studies reveal that proper telomere architecture is required for long-term proliferation capacity. Here we describe molecular mechanisms that protect and maintain chromosome ends and discuss why Arabidopsis is emerging as a powerful new model for elucidating fundamental aspects of telomere biology.     

Wiedemann‐Rautenstrauch (neonatal progeroid) syndrome: New case with normal telomere length in skin fibroblasts

Wiedemann‐Rautenstrauch (neonatal progeroid) syndrome is an autosomal recessive condition with characteristic appearance of premature aging present at birth (aged face, natal teeth, and wrinkled skin). Other features of the syndrome are generalized lipoatrophy with specific fat accumulation in the lateral suprabuttock region, hypotrichosis, macrocephaly (pseudohydrocephalus), and mental retardation. We report on a new case that demonstrates all typical features of the syndrome. The girl is now 16 years and 10 months old and has had follow‐up from birth. We measured terminal restriction fragment (TRF) length to evaluate whether the patient's premature aging process is accompanied by shortening of telomere length in her cultured fibroblasts. Mean TRF of 13.5 kb found in our patient's fibroblasts is not shortened as compared to that of normal fibroblasts. Our results differ from those observed in Hutchinson‐Gilford progeria. © 2001 Wiley‐Liss, Inc.     

Cytogenetics of a new cytotype of African Mus (subgenus Nannomys) minutoides (Rodentia, Muridae) from Kenya: C- and G- banding and distribution of (TTAGGG) n telomeric sequences

We present the results of a cytogenetic study on Mus (Nannomys) minutoides from Kenya by means of C- and G- banding and in-situ fluorescence hybridization (FISH) to localize the telomeric sequences. The karyotype is characterized by the occurrence of several Rb chromosomes Rb(1.X), Rb(1.Y). Rb(2.17), Rb(3.13), Rb(4.10), Rb(5.11), Rb(6.7), Rb(8.12), not previously described for this species. This finding suggests a high level of chromosomal diversification, which means it is possible to consider this cytotype as a new, well-differentiated, chromosomal lineage within the subgenus. The C-banding of the metaphases illustrated conspicuous blocks of centromeric heterochromatin at the paracentromeric regions of all telocentric chromosomes. Centromeric heterochromatin is not visible on all biarmed chromosomes. Following hybridization with telomeric probes, bright interstitial telomeric sequence (ITS) fluorescence signals are evident at the pericentromeric area of all Rb chromosomes, with the exception of Rb(2.17). Considering the localization of the C-positive heterochromatin and of the telomeric sequences, the events leading to the Kenyan cytotype from an all-telocentric condition probably included two steps: first, fusion without loss of heterochromatin and pericentromeric telomeric sequences; second, the reduction of the C-positive satellite DNA followed by the amplification of telomeric sequences in the C-negative paracentromeric region of Rb chromosomes. The presence of a single Rb(2.17) without ITS indicates possible variations of this mechanism.     

Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.     

Unusual distribution pattern of telomeric repeats in the shrews <Emphasis Type="Italic">Sorex araneus</Emphasis> and <Emphasis Type="Italic">Sorex granarius</Emphasis>

Sorex araneus and Sorex granarius are sibling species within the Sorex araneus group with karyotypes composed of almost identical chromosome arms. S. granarius has a largely acrocentric karyotype, while, in S. araneus, various of these acrocentrics have combined together by Robertsonian (Rb) fusions to form metacentrics, with the numbers and types of metacentrics differing between chromosomal races. Our studies on telomeric sequences in S. araneus and S. granarius revealed differences between chromosomes and between species. In S. araneus (the Novosibirsk race), hybridization signals were present on the telomeres of all the chromosomes after FISH with a PCR-generated telomeric probe. In addition, hybridization signals were observed at high frequencies in the pericentric regions of some but not all metacentrics formed by Rb fusion. There were fewer signals on those metacentrics formed earlier in the evolution of S. araneus. This suggests that S. araneus chromosomes retain at least some telomeric repeats during Rb fusion, but that these repeats are lost or modified over time. These results are critical for the interpretation of the well-studied hybrid zones between chromosomal races of S. araneus, given that Rb fission has been postulated in such hybrid zones and that the likelihood of Rb fission will relate to presence/absence of telomeric sequences at the centromeres of metacentrics. In S. granarius, there were strong signals at the proximal (centromeric) telomeres of the acrocentrics after FISH with a DNA telomeric probe. FISH with a PNA telomeric probe on S. granarius acrocentrics showed that the proximal telomeres were 213 kb on average, while the length of the distal telomeres was 3.8 kb on average. Two-colour FISH, using a telomeric DNA probe and a microdissected probe generated from the pericentric regions of the S. granarius chromosomes a and b, revealed regions on distinct chromatin fibres where telomeric and microdissected probes were colocalized or localized sequentially. The proximal telomeres of S. granarius are highly unusual both in their large size and their heterogeneous structure relative to the telomeres of other mammals.