用原位杂交法定位猪乳铁蛋白基因于染色体2q12

本研究以非放射性标记的猪乳铁蛋白(Porcine Lactoferrin简称PLF)cDNA 为探针,通过染色体原位杂交法,对PLF基因进行了染色体定位。实验中采用金胶抗体技术并结合使用银增强系统,提高了方法的灵敏度。利用染色体的组型分析,对杂交点的分布进行统计学分析。52%(26/50)的分裂相在第2号染色体具银粒分布,实验结果表明：PLF基因定位于猪2号染色体2q¹²区域。     

Localization of An Exogenous GH Gene on Chromosomes of Transgenic Pig by in situ Hybridizaiton

近些年来随着原位杂交技术的不断改进,该技术已广泛用于染色体的基因定位。非放射性标记探针的应用使基因定位变得更加简单易行,从而有可能对动物的转基因进行定位研究。本文首次采用胶体金标记药盒（Ａｎｔｉｄｉｇｏｘｉｇｅｎｉｎｇｏｌｄ）和银加强试剂（Ｓｉｌｖｅｒｅｎｈａｎｃｅｍｅｎｔｒｅａｇｅｎｔｓ）的非同位素原位杂交技术对转基因猪外源基因进行了定位研究。如Ｆｉｇ．１所示：表达质粒ｐＳＭＴＰＧＨ含有载体ｐＵＣ１９,羊启动子ＭＴ０１１和猪生长激素ＰＧＨ基因。选５头带有ｐＳＭＴＰＧＨ的转基因猪,分别制备含有染色体ＤＮＡ的杂交膜。用ＢｇｌＩＩ和ＳｍａＩ对ｐＳＭＴＰＧＨ进行完全酶切,收集０．９Ｋｂ片段作为探针,以ｄｉｇ１１ｄＵＴＰ进行标记。探针与ＤＮＡ杂交后,用Ａｎｔｉｄｉｇｏｘｉｇｅｎｉｎｇｏｌｄ和Ｓｉｌｖｅｒｅｎｈａｎｃｅｍｅｎｔｒｅａｇｅｎｔｓ进行显色反应。胰酶法Ｇ—显带后,用光学显微镜检查。选择分散相良好、显影银颗粒清楚的玻片进行摄影记录（Ｆｉｇ．２）。对染色体上的显影银颗粒进行统计分析,参照家猪的染色体标准带型,确定外源ＰＧＨ基因整合位点。Ｆｉｇ．３为４１０４号转基因猪染色体上的银颗粒分布情况。对５头     

急性早幼粒细胞白血病（APL）中t（15;17）染色体易位的分子机制研究

对急性早幼粒细胞白血病中ｔ（１５;１７）染色体易位的分子生物学研究显示,１７号染色体上的维甲酸受体α（ＲＡＲＡ）基因与１５号染色体上的ＰＭＬ基因并置,并产生ＰＭＬ－ＲＡＲＡ融合基因。我们以前的工作证明ＡＰＬ患者中ＰＭＬ基因断裂点集中于２个限区域,即ＰＭＬ－ｂｃｒ１和ＰＭＬ－ｂｃｒ ２,二者相距约１０ｋｂ。本文确定了ＰＭＬ－ｂｃｒ １的ＤＮＡ顺序,并确定了一例ＡＰＬ患者染色体相互易位接合部的基本结构     

染色体定位两个与玉米大斑病抗性基因Ht1连锁的RFLP标记

以玉米（ＺｅａｍａｙｓＬ．）黄早四自交系为材料,采用生物素标记的染色体原位杂交技术,对与大斑病抗性基因Ｈｔ１紧密连锁的两侧两个ＲＦＬＰ标记ｕｍｃ２２和ｕｍｃ１２２进行了定位。结果表明,ｕｍｃ２２和ｕｍｃ１２２探针都同时与第２号和第７号染色体杂交,而且,ｕｍｃ２２也和第４号染色体杂交。这些结果反映了这两个ＲＦＬＰ标记的二重或三重分布。两探针平均信号检出率分别为２０４２％和１４４９％。ｕｍｃ２２和ｕｍｃ１２２在第２号染色体长臂上杂交信号与着丝粒的百分距离分别为５８３６±３．１９和６１０２±４３２;在第７号染色体长臂上杂交信号与着丝粒的百分距离分别为４４７０±２．１１和４５１９±２２７。这些结果表明,ｕｍｃ２２和ｕｍｃ１２２在第２号染色体上的物理图的相对位置与遗传图的相对位置相符,彼此间都相距很近,两个标记在第２号和第７号染色体的重复是连在一起发生的。由此推断,Ｈｔ１除位于第２号染色体ｕｍｃ２２和ｕｍｃ１２２之间的位置外,在第７号染色体这两个标记之间的区域也应该存在它的同源序列     

水稻亚铁胁迫诱导ADH的基因定位及其遗传分析

籼稻品种ＩＲ６４与粳稻品种Ａｚｕｃｅｎａ及其ＤＨ群体１３５个系用于进行Ｆｅ＾２＋胁迫（２５０ｍｇ／Ｌ,ｐＨ４．５）及对照实验,对处理及对照条件下的ＡＤＨ进行基因定位及遗传分析,结果表明,在Ｆｅ＾２＋胁迫条件下,ＡＤＨ酶的活性大大提高,群体在Ｆｅ＾２＋胁迫条件下,表现低值的超亲现象,分布偏向ＩＲ６４,单标记分析和最大似然区间作图结果表明,Ｆｅ＾２＋胁迫条件下,１１号染色体上紧密连锁的３个标记位点ＲＧ     

应用ＣＡＴＳ法分离和鉴定猪ＧＦＡＰ基因的研究

根据比较锚定序列宗踪（ＣＡＴＳ）法,选择人和小鼠胶质细胞原纤维酸性蛋白（ＧＦＡＰ）基因的同源区域设计引,用ＰＣＲ方法从二花脸猪基因组中分离到４１２bp的基因片段,经与基因资料库中已训功能基因的同源性比较,该片段可鉴定为猪的ＧＦＡＰ基因,利用猪－啮齿类体细胞杂克隆板将ＧＦＡＰ基因定位于猪１２号染色体１２p11-(2/3)P13区域。     

水稻光敏素基因(phyA)和-1,5-二磷酸核酮糖羧化酶/加氧酶小亚基基因(rbcS)的染色体定位

以生物素标记的水稻单拷贝光敏素基因（ｐｈｙＡ） 和１ ,５二磷酸核酮糖羧化酶／ 加氧酶小亚基基因（ｒｂｃＳ） 的基因组克隆为探针,其大小分别为６ ．６ 和１ ．１ ｋｂ ,通过原位杂交技术将它们分别定位到水稻染色体上。ｐｈｙＡ 和ｒｂｃＳ基因的检出率分别为２９ ．７９ ％ 和２１ ．５６ ％ 。ｐｈｙＡ在第３ 染色体上有３ 个座位：长臂近着丝粒、短臂末端、长臂中部。ｒｂｃＳ分别定位于第７ 染色体长臂近着丝粒（８ ．６２ ％ ） 、第５ 染色体长臂末端、第６ 染色体长臂距着丝粒近２／３ 处。此外,对信号转导相关基因定位的意义,水稻染色体的准确识别、功能基因在染色体上的分布及位置意义等也进行了讨论。     

利用AFLP遗传连锁图定位大麦苗期对叶锈病的部分抗性基因

借助大麦染色体ＡＦＬＰ标记遗传连锁图和ＭａｐＱＴＬＶ３．０作图软件,对大麦叶病的数量抗性基因进行了定位分析,明确了大麦部分抗性品种Ｖａｄａ对叶锈病的潜育期由分别位于染色体１、２、６、７上离短臂末端７９ｃＭ、１８６ｃＭ、５８ｃＭ和１１７ｃＭ处的４个数量抗性基因所控制。     

转基因猪染色体原位杂交外源生长激素基因定位

近些年来随着原位杂交技术的不断改进,该技术已广泛用于染色体的基因定位。非放射性标记探针的应用使基因定位变得更加简单易行,从而有可能对动物的转基因进行定位研究。本文首次采用胶体金标记药盒（Anti-digoxigenin-gold)和银加强试剂（Silver enhance-ment reagents)的非同位素原位杂交技术对转基因猪外基因进行了定位研究。如Fig.1所示：表达质粒pSMTPGH含有载体pUC19,羊启动子MT011和猪生长激素PGH基因。选5头带有pSMTPGH的转基因猪,分别制备含有染色体DNA的杂交膜。用BglII和Smai对pSMTPGH进行完全酶切,收集0．9kb片段作为探针,以dig-11-dUTP进行标记。探针与DNA杂交后,用光学显微镜检查。选择分散良好、显影银颗粒清楚的玻片进行摄影记录（Fig.2)。对染色体上的显影银颗粒进行统计分析,参照家猪的染色体标准带型,确定外源PGH基因整合位点。Fig.3为4104号转基因猪染色体上的银颗粒分布情况。对5头转基因猪外源PGH基因定位的结果见Table1。探针的合理设计是外源基因定位研究成功的关键。本实验所用探针必须地与外源PGH基因杂交,而不受内源PGH基因的影响。我们设计的探针符合这一要求。采用dig11－dUTP标记探针,抗体金显色,银加强试剂放大杂交信号,在光学显微镜下可以直接观察杂交位点处的显影银颗粒,但于对实验进行统计分析。估计数据表明：转基因猪的外源PGH基因随机整合在所有染色体上,但在13号染色体上的机率略高。     

蝮蛇毒碱性磷脂酶A_2基因的克隆

从蝮蛇毒腺中抽提总ＲＮＡ．利用人工合成寡核苷酸引物作逆转录,以ｃＤＮＡ为模板进行体外扩增,获得磷脂酶Ａ２（简称ＰＬＡ２）基因,克隆至ｐＢＳ－ｋｓ载体中。通过对３个碱性ＰＬＡ２（简称ＢＰＬＡ２）基因单独克隆分别作ＤＮＡ全序列分析,推导ｐｒｏ－ＢＰＬＡ２由１３８个氨基酸残基构成,与已测定的部分氨基酸序列比较,基本相符。该基因成功的克隆,不仅推导出ＢＰＬＡ２的蛋白质全序列,也为进一步开展蛇毒功能肽蛋白质工程的研究工作打下了良好的基础。     

Localization of the KRAS2 oncogene in the domestic rabbit (Oryctolagus cuniculus)

The KRAS2 gene was localized in the rabbit by chromosomal in situ hybridization, using a 3H-labeled human cDNA probe. There were 201 silver grains on 144 metaphase spreads; 12.9% of the grains resided on chromosome 16, and 54% of these grains were located close to the centromere at 16p11----q11. Statistical analysis indicated that labeling at this region represents a significant deviation from a random distribution and thus provided evidence for the assignment of KRAS2 to 16p11----q11. In addition to the predominant labeling site on 16, there was a positive signal at the telomere of 9q, possibly representing a sequence of another member of the ras family. Our assignment of KRAS2 to rabbit chromosome 16 strengthens the argument that a fragment on this chromosome is homologous to one on human chromosome 12, which bears the KRAS2 locus.     

用原位分子杂交技术定位牛生长激素基因于5号染色体

本工作利用放射性标记的ｂＧＨ基因（３．０ｋｂ）为探针,通过原位杂交定位牛生长激素基因于染色体５ｑ２２－２６内。该结果与以前的ｂＧＨ基因定位的结果不同,讨论了基因探针、基因定位方法等方面与定位准确性的关系。     

Chromosomal localization of a bovine male specific probe

The B.C.1.2. probe, a fairly repeated sequence of the bovine male genome, was located by in situ hybridization on cattle karyotype. Of a total of 711 silver grains, 139 (19.5%) clustered on chromosome Y and about 71% of the grains were located on the short arm. This Y specific bovine male probe could be useful for routine embryo sexing in connection with embryo transfer.     

Adult muscle sodium channel alpha-subunit is a gene candidate for malignant hyperthermia susceptibility.

Human myosin light chain-2 (MYL2) is an important protein involved in the regulation of myosin ATPase activity in smooth muscle. In cardiac muscle, the precise role of MYL2 is not well understood; however, an increase in ventricular MYL2 is observed during myocardial hypertrophy in cardiac patients with valve stenosis. The chromosomal location of the gene coding for MYL2 was identified using a cloned cDNA for human MYL2. Southern blot analysis of DNA from a human/rodent somatic cell hybrid mapping panel showed that the BamHI fragment that hybridized with this cDNA probe was concordant with chromosome 12. The 768-bp cDNA was hybridized to human metaphase chromosomes. The results revealed a significant clustering of silver grains over chromosome 12 bands q23-q24.3, indicating that the gene coding for MYL2 is located in this region.     

人14p＋标记染色体的分子细胞遗传学研究

一例23岁女性患者因近五、六年来出现胡须、四肢多毛及偶有月经不规则而就诊。细胞遗传学检查发现一个短臂明显增大的亚中着丝粒的14号标记染色体14p ·p 区域GTG显带呈浅染,C-带暗染,都呈均匀的染色区。硝酸银染色在p 远侧端显现一个Ag-NOR,其大小与正常近端着丝粒染色体的无明显差异。应用~3H标记的7.3 kb长的rRNA基因探针进行染色体原位杂交,自显影银颗粒沿整个p 区域分布,p 上的银颗粒数是正常近端着丝粒染色体短臂上银颗粒平均数的5倍。这些结果排除了Y或其他染色体参加的重排形成p 的可能性,并表明Ag-NOR的大小或NOR的数目并不一定与rRNA基因的数量成正比。研究Dp 或Gp 类型的染色体变异,对了解人二倍体细胞内rRNA基因表达的调控有重要意义。     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

Chromosomal localization of leukocyte interferon gene in the pig (Sus scrofa domestica L.) by in situ hybridization

Using as a probe pig genomic DNA, including the complete interferon alpha gene, we have mapped the leukocyte interferon gene on pig chromosome 1 by in situ hybridization. A total of 196 silver grains were noted on the 106 metaphases scored: 31% of the grains were observed on chromosome 1, and 67% of these were localized in the region 1q2.2----q2.7.     

用染色体原位杂交技术定位RFLP探针Fr.3-42于16p13

限制性片段长度多态性(RFLP)探针Fr.3-42(第九届人类基因定位国际会议编号D1(?)S21)为一长1.9kb的人类单拷贝EcoRI/HindⅢ片段,本实验采用染色体原位杂交方法,将该探针定位于16号染色体短臂末端(p13)。在另一研究中已证实Fr.3-42与人α-珠蛋白基因紧密连锁。     

Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization.

Prealbumin is a serum protein which plays an important role in plasma transport of retinol and thyroxine. The accumulation of a variant prealbumin is associated with a hereditary disorder, familial amyloidotic polyneuropathy (FAP). In situ hybridization with a mouse prealbumin gene cDNA probe was carried out in mouse fibroblasts. Analysis of 114 R-banded metaphases showed that 13% of the total grains were located on chromosome 4 and 46% of the grains on this chromosome were in the region C6-D1. Linkage and syntenic group analysis showed that the prealbumin gene (Ttr) is located between two syntenic groups on mouse chromosome 4, which corresponded to two syntenic groups present on human chromosomes 1 and 9.