应用荧光原位杂交(FISH)技术研究 黑叶猴染色体易位

本文应用染色体荧光原位杂交(FISH)技术,利用人9号和14号染色体特异探针,对深低温冻存和长期传代的黑叶猴细胞株染色体畸变进行了分析。确定在长期冻存和传代过程中,一些黑叶猴细胞在No.12和No.17染色体之间发生了易位,一条 No.17染色体发生断裂,断裂点在17q13,断裂片段17q13-17qter易位到一条 No.12染色体长臂末端,形成一条小的中着丝粒的和一条具较长长臂的衍生染色体即 der(17) 和 der(12)。结果表明,荧光原位杂交技术用人染色体特异探针不仅能检测出人类染色体畸变,也能有效地检测灵长类动物染色体畸变。 Abstract　In this paper,the chromosome aberration of long-term cryopreserved and subcultured Francois' monkey (Semnopithecus francoisi) cell line(KCB 92008) was analyzed by fluoresence in situ hybridizaton (FISH) using human 9 and 14 chromosome DNA probes. After compared the hybridization pattern with the G-banding pattern on the same metaphase,a translocation between Nos.12 and 17 chromosomes was identified. In some Francois'monkey cells,one of chromosome No.17 was broken into two at the breakpoint 17q13,the segment(17q13-17qter) without centromere transfered to the long arm terminal of one chromosome No.12. Thus,two derivant chromosomes der(12) and der(17) were formed,the long arm of der(12) was longer than the normal partner,while the long arm of der(17) was shorter than the normal one. The result indicated that the technique of FISH using human whole chromosome probes was not only a powerful tool to detect human chromosome rearrangements,but also a usefulmethod to study the primate chromosome aberration.     

玉米cyclinⅢ基因的染色体原位杂交物理定位

本文首次报道了玉米低拷贝基因 cyclinⅢ(B-类)生物素标记的染色体原位杂交定位结果。供试探针为该基因的cDNA克隆,其长度仅为1.6kb。结果表明, 探针的信号分布在第6染色体短臂和第9染色体长臂,与着丝粒的百分距离分别为70.05±3.31和86.86±1.64,检出率分别为8.29%和6.83%。文中对基因的物理位置与功能间的关系等作了讨论。 Abstract:A biotin-labelled in situ hybridization technique was used to physically map a low copy gene cyclinIII in maize.The cDNA clone was 1.7kb in size.The probe was hybridized onto the short arm of chromosome 6 and the long arm of chromosome 9.The percent distances from centromere to detection site were 70.05±3.31 and 86.86±1.64 respetively.The detection rates of in situ hybridization were 8.29 and 6.83 respectively,The relationship between the position and function of the genes is discussed in this paper.     

染色体微切割、微分离、微克隆技术及其研究进展

自1981年染色体微切割及微克隆技术创建以来,该技术已广泛应用于人类及动植物遗传学、医学、进化学等研究领域,主要包括构建特定染色体或染色体区域的DNA文库、制备染色体描绘探针池以研究染色体重排和染色体进化等。本文对该技术的产生、发展及某些研究进展作一综述。 Abstract: Since chromosome microdissection and microcloning technique was developed in 1981,it has been wide ly used in genetics,medicine,evolution and other fields,mainly in establishing chromosome or chromosone specific region DNA libraries,preparing chromosome painting probe pools to study chromosome rearrangement and evolution.In the paper,the development and research progress of the technique are discussed.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

应用染色体涂染技术分析两例染色体结构异常 Analysis of the Structure of Abnormal Karyotypes by Using Chromosome Painting

为了确定两例细胞遗传学提示染色体结构异常的核型,应用通过显微切割技 术构建的人类18号和7号染色体探针池,分别对这两例病例的中期分裂相进行染色体涂染,结合显带染色体,确定两者核型分别为46,XY,t(3;18) (q12;q21)和46,XX,dir ins(1;7)(p3104;q34q36)。染色体涂染技术是染色体显带技术的重要补充和发展,为染色体结构异常提供了一种直观、准确的检测手段,在遗传咨询和产前诊断方面有重要作用。 Abstract:In this study,chromosome painting technique was performed to analyse the abnormal karyotypes of two carriers.Chromosome 18 and 7 specific libraries,which were generated by chromosome microdissection technique,were used as probe pools to hybridize the carriers metaphase chromosomes respectively.Unlabled human genomic DNA was used to inhibit the hybridization of sequences in the library that bind to mutiple chromosomes.Structure abnormality was detected clearly in metaphase.Combined with the banding chromosomes,we concluded that their karytypes were 46,XY,t(3;18)(q12;q21)and 46,XX,dir ins(1;7)(p3104;q34q36).Chromosome painting,as a direct and concise method in analysing chromosome structure abnormality,is an important complement and development of chromosome banding technique,and has important application in genetic counselling and prenatal diagnosis.     

组织凝集素在人染色体上的基因定位

本文用非放射性DIG标记组织凝集素基因探针研究其在人染色体上的基因定 位,首先对基因原位杂交条件进行了一系列研究。结果表明采用染色体温和变性、基因 片段探针等为较好的基因原位杂交条件,通过135个细胞中期相的分析结果显示, 杂交阳 性率为20%,本底为2.81,杂交点分布峰位于3q12-13。 Abstract:The localization of sarcolectin gene on human chromosome is studied.A modified system of in situ hybridization was developed.A specific signal was generated by employing a nonradioactive DIG-labelled gene fragment as a probe in combination with the hybridization buffer containing no dextran sulphate and softly denatured chromosomes.Using this system,unique sequence for sarcolectin as small as 0.65kb was detectable and observed at 3q12-13.Of 135 metaphases examined,27(20%) had at least one grain deposited on 3q12-13,and the backgrand was 2.81.     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

大鳞副泥鳅ZZ/ZW型性别决定的细胞遗传学证据

大鳞副泥鳅是鲤形目、鳅科的鱼类。其2n数为48,核型组成为12m+4sm+32 t(雄性),11m+5sm+32t(雌性)。根据银染带和C带特征分析,证实大鳞副泥鳅为ZZ/ZW型性别决定。Z染色体为中部着丝粒染色体,在其长臂端部有Ag -NOR存在。 W染色体为亚中部着丝粒染色体,在其长臂末端也有Ag-NOR存在,同时还有一深染的居间C带,这是W染色体独有的带纹特征。 Abstract:Paramisgurnus dabryanus belongs to Cypriniformes,Cobitidae.Its 2n is 48.The karyotype formula is 12m+4sm+32t(in male),11m+5sm+32t(in female).According to the Ag-NORs band and C-band patterns,we consider that its sex determination is of ZZ/ZW type.The Z chromosome is a metacentric one with Ag-NORs located on its arm end.The W chromosome is a submetacentric with Ag-NORs located on the terminal of its long arm.There is a darkly stained C-band on the long arm of W chromosome.This band is a characteric of the W chromosome.     

一个水稻重复序列的分析与定位

在利用PCR简并引物扩增水稻NBS-LRR类抗病基因同源序列的研究中,克隆了一个大小为560 bp左右的重复序列,命名为DH17。序列分析和同源性比较发现,该序列包含352 bp的重复单位,与已报道的OS48和TrsA等重复单位序列进行比较,差异多低于5%, 具有很高的同源性,因此为同一重复序列家族。分子杂交表明,该序列在籼型品种"窄叶青8号"（ZYQ8）中以大量的串联拷贝存在,拷贝数显著高于粳型品种"京系17"（JX17）。利用ZYQ8和JX17组配的DH群体,通过 RFLP分析,直接将DH17的大量串联拷贝定位于ZYQ8的12号染色体长臂末端区域。 Abstract:A repeated sequence with a length of 560 bp,termed as DH17,was obtained during PCR amplification of rice NBS-LRR homologues.A repeated unit of 352 bp in the DH17 fragment was revealed through sequence analysis and comparison,which has a high homology with the known sequences of OS48 and TrsA,and belongs to the same repeat family.Southern hybridization displayed that there are higher DH17 copies in the genome of an indica variety,ZYQ8,than that in the genome of japonica variety,JX17.The tandom repeated DH17 sequence was mapped on the long arm end of chromosome 12 through RFLP analysis of a double haploid population derived from ZYQ8 and JX17 using DH17 as a probe.     

Prevalence of antibody reaction with cercopithecine herpesvirus 1 antigen in Macaca cyclopis, Macaca fascicularis, and Papio anubis in Taiwan

BACKGROUND AND METHODS: A total of 284 non-human primate sera were collected between December 2004 and September 2005 and tested by a commercially available dot immunobinding assay for the antibodies to cercopithecine herpesvirus 1, an alphaherpesvirus with high mortality for infected humans. RESULTS: Seropositive rates were 58% among non-human primates from animal shelters and 38% among those from zoos and academic institutes. Positive reactors were found in three species, the Formosan macaque (Macaca cyclopis; 57%), the cynomolgus macaque (Macaca fascicularis; 11%) and the olive baboon (Papio anubis; 68%). CONCLUSIONS: Our results showed that natural infection by cercopithecine herpesvirus 1 in Formosan macaques was highly prevalent, and to a certain extent reflected the situation of the wild populations in Taiwan. The findings raised the issues of zoonotic public health and the occupational health of primate workers. High positive rate in olive baboons was also found, although, it cannot be ruled out that the positivity was due to cross-reactivity between cercopithecine herpesvirus 1 and other herpesviruses.     

一种改进的阴茎电刺激采精法和猕猴藏酋猴及熊猴的采精及其精液特征的初步研究

本文报道了一种改进的阴茎电刺激采精法,用脱脂棉和铝箔作为电极,以避免直接用金属电极可能对阴茎的损伤,并运用这一方法对猕猴（Ｍａｃａｃａｍｕｌａｔｔａ）、藏酋猴（Ｍ．ｔｈｉｂｅｔａｎａ）和熊猴（Ｍ．ａｓｓａｍｅｎｓｉｓ）进行了电刺激采精及其精液特征研究。电刺激采精模式为连续刺激和间断刺激方式。在采精过程中没有发生阴茎损伤。对初次接受电刺激采精的动物以间断刺激模式效果较好。猕猴、藏酋猴和熊猴的射精体积分别为２．０±０．１、６．３±１．１和３．２±０．６ｍｌ;液化体积分别为０．７±０．６、２．１±０．４和１．７±０．３ｍｌ;精子浓度分别为１２．６±１．２×１０~８、４５．６±５．６×１０~８和１１．５±０．９×１０~８／ｍｌ。３种动物精液的液化率分别为：猕猴３６．２±０．９％,藏酋猴３４．０±１．４％;熊猴５１．８±１．２％。３种动物的精子总数与射精体积和凝块体积没有相关性（ｒ~２＝０．０７９;０．０１６;０．０９４和ｒ~２＝０．０６４;０．０２０;０．０７２）。上述结果表明：１）改进的阴茎电刺激采精法适用于猕猴,特别是阴茎表面较为粗糙的藏酋猴和熊猴;２）藏酋猴的射精体积和精子总数是迄今已报道的非人灵长类中最大的,可能     

食蟹猴和熊猴杂种F1的细胞遗传学研究

本工作采用染色体显带、睾丸组织学切片以及低渗铺张－硝酸银染色等方法较为详细地研究了食蟹猴和（Ｍ．ｆａｓｃｉｃｕｌａｒｉｓ）和熊猴（Ｍ．ａｓｓａｍｅｎｓｉｓ）种间杂种Ｆ１体细胞染色体的Ｇ带、Ｃ带、Ａｇ－ＮＯＲ、精母细胞联合复合体的结构、形态和行为以及精子发生。结果表明：（１）杂种亲本的染色体组具有高度的同源性;（２）杂种的精子发生过程完全正常;（３）食蟹猴和熊猴的种间生植屏障可能主要是生态隔离。此外     

Leptin, adiposity, and testosterone in captive male macaques

Leptin is considered to act as a signal relating somatic energetic status to the reproductive system. However, the nature of that signal and its relationship with male reproductive function across nonhuman primate species are unclear. We suggest that species-specific differences in leptin physiology may be related to the degree of environmental variation and variation in the importance of energy stores for male reproduction. In order to test the role of seasonality in species differences among nonhuman primates, we compared leptin, testosterone, and body composition in male rhesus (n = 69) and pig-tailed (n = 43) macaques. Despite having larger abdominal fat deposits, the rhesus macaques did not exhibit significantly higher leptin levels (rhesus, 2.21 +/- 0.43 ng/ml; pig-tailed, 2.12 +/- 0.39 ng/ml). Both species showed increases in leptin across adolescent, subadult, and adult age-groups (P = 0.036 for rhesus; P = 0.0003 for pig-tailed by ANCOVA). Testosterone was not significantly associated with leptin in either the rhesus (r = 0.039; P = 0.754) or pig-tailed (r = 0.2862; P = 0.066) samples. Comparison of leptin levels across the two species using univariate modeling procedures showed no significant age-group by abdominal fat interaction. These findings suggest little difference in leptin production between these two closely related species, despite the difference in breeding seasonality.     

Confirmation and location of the hybrid zone between wild populations of Macaca tonkeana and Macaca hecki in Central Sulawesi,Indonesia

Reports of hybridization between Macaca tonkeana and Macaca hecki were investigated in Central Sulawesi, Indonesia. We defined sets of morphological traits that were diagnostic for M. tonkeana and M. hecki and then located an areas where animals had intermediate or mosaic features. Hybridization as indicated by morphology was detected between M. tonkeana and M. hecki. The hybrid zone appeared to be strongly centered at the road that crosses the isthmus of Central Sulawesi from Tawaeli to Toboli. Macaques in this region were not morphologically uniform; animals from the western area of the Tawaeli–Toboli road resembled M. hecki, while animals from the eastern area resembled M. tonkeana. The hybrid zone was found to be smaller than previously thought, with maximum dimensions of approximately 15 and 7.5 km. Clines for diagnostic morphological features were broadly coincident, suggesting that the hybrid zone originated by secondary contact. Analysis of three museum specimens collected in 1916 provided evidence that the hybrid zone has been in existence since at least then. The narrow width of the hybrid zone, along with its age, suggested that some prezygotic or postzygotic barrier must exist to full introgression between M. tonkeana and M. hecki. Am. J. Primatol. 43:181–209, 1997. © 1997 Wiley-Liss, Inc.     

Some observations on psychosocial stressors,immunity, and individual differences in nonhuman primates

Many studies would appear to support an important relationship between behavior and the regulation of the immune response, at the core of the developing field of psychoneuroimmunology (PNI). However, wide variations are noted in the strength of this relationship, even for the same species under very similar circumstances. The present paper reviews work from our laboratory in both young and adult pigtail and bonnet macaques describing the ranges of variability in both behavioral and immunological responses to social separation and social conflict. It is shown that the magnitude of immune changes that follow a social stressor often covary with the magnitude of the behavioral changes that are observed. Second, there is support for the observation that there are specific behaviors such as social affiliation that may mitigate untoward behavioral and immunological consequences of social stressors. Finally, studies are reviewed which suggest that assessment of autonomic reactivity as reflected in cardiovascular regulation might provide an important clue to the relative risk for immune modulation under challenge. © 1996 Wiley-Liss, Inc.     

Wounding patterns in three species of captive macaques

The incidence of wounding in captive groups of rhesus (Macaca mulatta), pigtail (M. nemestrina), and stumptail (M. arctoides) macaques was studied for 21 months. Groups were monitored daily for evidence of wounding. Wounded animals were captured, treated by veterinary staff, and returned following recovery. Records were kept on the age, sex, and species of the recipient, along with the type and location of wound. In each species of macaque, adult males incurred the highest frequency of wounds and multiple wounds of any age-sex class. This contrasted with previously reported behavioral data indicating low frequencies of aggression received by adult males, especially contact aggression and bites. These discrepancies indicate wounding frequencies do not necessarily correspond with behavioral measures of aggression. Inhibition of aggression directed toward infants and the selective avoidance of bites directed to vital body regions were presented as possible mechanisms that modify intragroup aggression. Increased wounding in the birth season under captive conditions suggests that the pattern of increased wounding reported during the breeding season under freeranging conditions may reflect xenophobic responses to immigrating males, rather than direct male-male competition for estrous females.     

Ultrasonic assessment of pregnancy and fetal development in three species of macaque monkeys

Serial ultrasonic assessments of gestational sac (GS) and fetal biparietal diameter (BPD) were performed in the Japanese monkey (Macaca fuscata fuscata), rhesus monkey (Macaca mulatta), and crab-eating monkey (Macaca fascicularis). In the Japanese monkey, GS increased linearly for 3-8 weeks, whereas BPD increased in a linear-quadratic manner over 8 weeks to term. Ultrasonic assessments of spontaneously aborted fetuses with BPD growth retardation and diagnosis of a pelvic chocolate cyst also were reported.     

滇金丝猴,藏酋猴和毛耳猴下颌骨的异速生长

本文对滇金丝猴(Rhinopithecus bieti)、藏酋猴(Macaca thibetana)和毛耳猴(Macacamulatta lasiota)下颌骨的33项指标进行了测量.经分析,下颌骨的生长存在有部位间、左右间及类群间的差异。表明这些动物下颌骨形态因适应某种生态环境而可能存在某些相似性,但因其食物性质、取食行为、系统及个体发育过程中存在差异而出现不同的生长模式。