APPLICATION OF HIGH PRESSURE LIQUID CHROMATOGRAPHY AND MICROSEQUENCING METHODOLOGY IN THE STRUCTURAL ANALYSIS OF HUMAN HEMOGLOBIN VARIANTS

This paper reports the results of the analyses of 17 different Hb variants which were observed in 37 families. High pressure liquid chromatography (HPLC) was used to separate tryptic and thermolytic peptides of globin chains. The double coupling microsequencing technique can successfully be used for sequencing small or larger peptides.     

Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.     

A NEW SILENT MUTATION FOUND IN THE CHINESE PAH LOCUS AND ITS ROLE IN THE PRENATAL DIAGNOSIS OF PHENYLKETONURIA

A silent mutation or sequence polymorphism, A to T substitution at codon 399 in exon11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The fre-quencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09,respectively, based on the analyses of 100 normal individuals and 39 PKU patients usingDNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridizationmethods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagno-sis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPslinkage analysis, was prenatally diagnosed with this genetic marker.     

A STUDY ON CHINESE PHENYLALANINE HYDROXYLASE GENE RESTRICTION SITE POLYMORPHISM

Human phenylalanine hydroxylase (PAH) cDNA was applied as a hybridization probe to analyzing the following 8 restriction fragment length polymorphisms (RFLP) in the PAH genes of 80 normal and 28 phenylketonuric Chinese patients: BgⅢ, 3.6kb/1.7kb; EcoRI, 17kb/11kb; EcoRV, 30kb/25kb; HindⅢ, 4.2kb/4.0kb; MspIa, 23kb/19kb; MspIb, 4.0kb/2.2kb; PvuIIa, 19kb/6.0kb and PvuIIb, 11.5kb/9.1kb. The frequencies of the above RFLP in normal Chinese and PKU patients are: 0.13, 0.83, 0.77, 0.81, 0.12, 0.04, 0.70, 0.10 and 0.12, 0.93, 0.89, 0.81, 0.04, 0, 0.69, 0.04, respectively. This study reveals significant differences between the frequencies of individual RFLPs when these values are compared with those of the Caucasians. Finally, the detection of RFLPs in the PAH gene in the Chinese population will permit prenatal diagnosis of PKU in China.     

羟基脲(HU)治疗β-地中海贫血的研究——HU对珠蛋白基因表达的影响

本文应用RT-PCR/竞争性PCR测定mRNA相对含量和绝对含量的方法,以及微量珠蛋白肽链生物合成技术,系统地观察了β-地中海贫血病人HU治疗前后珠蛋白基因表达的变化,首次发现HU可使一些病人的β-珠蛋白基因的表达明显增高,两例经HU治疗两年以上的病人因其β-珠蛋白mRNA和β-珠蛋白肽链合成的显著增加,导致其血液学上的明显改善和临床症状的好转。     

高压液相层析和微量顺序测定法在人类血红蛋白变种结构分析中的应用

本文报道在37个家系中所观察到的17种不同类型血红蛋白分析结果。所采用的方法是用高压液相层析(HPLC)来分离珠蛋白链的胰蛋白酶消化产物或肽的嗜热菌酶消化产物。HPLC具有快速分离及需用样品少的优点,而且层析谱的重复性好。对于小肽段或较大肽段的氨基酸顺序测定应用微量双偶合顺序测定技术是成功的。     

在中国人PAH基因中发现一种新的中性突变以及它在苯丙酮尿症产前诊断中的作用

本文在对一例中国PKU患者的PAH基因进行碱基顺序分析时发现了一种新的中性突变或顺序多态位点:11外显子第399密码子GTA(Val)→GTT(Val)。应用PCR体外扩增和寡核苷酸杂交技术对100个正常人和39个PKU患者进行分析,发现此顺序多态性在正常和异常PAH基因中的分布频率分别为0.005和0.090,存在一定的差异性。该顺序多态可作为一种“遗传标记”成功地对一例不能用RFLP连锁分析作完全诊断的PKU高危胎儿进行了产前诊断。     

应用mRNA/PCR技术研究中国人常见的一种β-地中海贫血基因(IVS-Ⅱnt.654 C→T)的剪接缺陷

应用扩增cDNA的直接序列测定,分析了中国人一种常见的β-地中海贫血突变类型(ⅣS-Ⅱ nt. 654 C→T)的转录产物和mRNA的剪接缺陷,结果表明这一突变基因除了导致β-珠蛋白m RNA的加工错误外,仍然产生少量正常剪接加工的mRNA。从而导致β~+地中海贫血。本文报道的方法为在转录水平上研究基因的表达,以及为遗传性疾病的分子缺陷提供简便和灵敏的新途径。     

应用PCR扩增牛SRY序列进行奶牛胚胎性别鉴定

本工作通过对牛SRY的直接测序,设计出牛SRY特异PCR引物,并应用PCR扩增奶牛胚胎的SRY特异序列来鉴定胚胎性别,经胚胎移植证实胚胎性别鉴定结果正确。     

中国人苯丙氨酸羟化酶基因的限制酶位点多态性研究

本文应用人类苯丙氨酸羟化酶(PAH)cDNA作杂交探针,分析了80例中国正常人和28例苯丙酮尿症(PKU)患者PAH基因的八个限制性内切酶多态性位点:Bgl Ⅱ 3.6kb/1.7kb,EcoRI 17kb/11kb,EcoRV 30kb/25kb,Hind Ⅲ 4.2kb/4.0kb,Msp Ⅰa 23kb/19kb,Msp Ⅰb 4.0kb/2.2kb,Pvu Ⅱa 19kb/6.0kb和Pvu Ⅱb 11.5kb/9.1kb。测得中国人正常PAH基因的上述限制酶位点多态性(RFLP)的发生频率分別为0.13,0.83,0.77,0.81,0,12,0,04,0.70和0.10;而缺陷PAH基因的上述RFLP发生频率分别为0.12,0.93,0.89,0.81,0.04,0,0.69和0.04。这表明中国人的PAH基因限制酶位点多态性和白种人的有较大差异。可以通过PAH基因的RFLP检查进行中国人PKU的产前诊断。