卵巢癌抗独特型单链抗体6B11ScFv／鼠HSP70融合蛋白复性条件的研究

目的探讨大肠杆菌表达的卵巢癌抗独特型单链抗体／小鼠热休克蛋白70（6811ScFv／mHSP70）融合蛋白包涵体变性、复性条件,以确定其最佳体外复性条件。方法大肠杆菌表达大量融合蛋白6811ScFv／mHSP70：①比较不同的裂解液（8mol／L尿素和6mol／L盐酸胍）对包涵体的裂解效率及其对复性蛋白活性的影响;②探索序贯稀释方法,以及氧化还原环境和复性液中不同浓度L-精氨酸对蛋白稀释复性的影响;③比较稀释复性和柱。卜复性对融合蛋白复性效率及活性的影响。采用Bradford法测定包涵体裂解液与复性后蛋白浓度。所有复性蛋白活性的检测均采用ELISA方法。结果以包涵体形式表达6811ScFv／mHSP70蛋白：①经6mol／L盐酸胍裂解包涵体的效率远高于8mol/L尿素,但前者复性所得蛋白活性低,6mol／L盐酸胍彻底裂解的包涵体经8mol／L尿素透析后再复性,复性效率显著提高;②序贯稀释方法可提高蛋白复性效率;加入0．5mol／L L-精氨酸可降低稀释复性过程中蛋白聚集物的产生,提高复性效率;加入还原型谷胱甘肽（GSH）与氧化型谷胱甘肽（GSSH）浓度比为1：5时,可提高复性后蛋白活性;③柱上复性可一步完成蛋白的复性和纯化,所得蛋白纯度较高,但复性效率和复性后蛋白活性较稀释复性低。结论本研究建立了6811ScFv／mHSP70融合蛋白的最佳复性条件：包涵体经6mol／L盐酸胍彻底裂解后,再经8mol／L尿素透析,然后进行序贯稀释复性,且复性液中加入0．5mol／L L-精氨酸和GSH：GSSH=1：5以提高复性效率和蛋白活性,为进一步研究该蛋白的功能奠定了基础。     

金黄色葡萄球菌 eap 基因的克隆与表达

 目的 构建携带 eap 基因的原核表达载体，诱导表达具有活性的重组 EAP 融合蛋白。 方法 PCR 法扩增金黄色葡萄球菌基因组 DNA，回收、 纯化的扩增产物与 pMD18-T 载体相连接得重组质粒 pMD18-T-EAP，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；未酶切组作为对照组重组质粒 pMD18-T-EAP 和 pET28a（+）表达载体分别用 Nde I 和 Xho I 限制性内切酶双酶切、连接，转化 E.coli BL21（DE3）感受态细胞，酶切鉴定；空载体作为对照组。用不同浓度（终浓度 1、2、4、8 mmol/L）和不同诱导时间（1、2、3、4、5、6 h）的异丙基-β-D-硫代半乳糖苷（IPTG）对阳性重组菌进行表达优化，分别取 E.coli上清液和沉淀做电泳分析。应用 MagneHis^TM 蛋白纯化系统纯化重组 EAP 融合蛋白，并通过薄层扫描测定蛋白质的浓度。 结果 所获 eap 基因与 GeneBank 的基因序列同源性 > 99%；氨基酸同源性达 100%。重组质粒经 IPTG 诱导，阳性重组菌转化子均有表达；当吸光度（A ）值等于 0.6 ~ 0.8 时，相对分子质量约 70 000 处出现目的蛋白条带。破碎的重组菌 pET28a-EAP上清液中目的蛋白条带较清楚，沉淀中几乎看不到。终浓度 1 mmol/L 为最佳蛋白表达工作浓度。IPTG 诱导 1 h 重组 EAP 融合蛋白有一定量的表达，随着时间的延长，表达量增加不明显，3 h 时的表达量达最高，之后，蛋白表达量变化不明显。表达的重组 EAP 融合蛋白含量占全菌体蛋白的 29.6%。 结论 成功地克隆和表达了金黄色葡萄球菌重组 EAP 融合蛋白，为进一步研究以 EAP 蛋白作为免疫原预防和治疗由金黄色葡萄球菌引起的疾病奠定基础。     

胰高血糖素衍生物在大肠杆菌的克隆表达和纯化

 目的 通过基因工程途径获得胰高血糖素衍生物。 方法 根据胰高血糖素基因及凝血酶酶切位点序列，分段合成引物行 PCR 扩增，以 PCR 扩增得到的胰高血糖素-甘氨酸基因序列和 pET-30a 质粒转化 E.coli DH5α，获得重组质粒 pET-G。将重组质粒 pET-G 转化至 E.coli BL21(DE3)，重组菌株命名为 E.coli BL21[pET-G]。以异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，对表达产物进行SDS-Tricine- PAGE[十二烷基硫酸钠-三（羟甲基）甲基甘氨酸-聚丙烯酰胺凝胶电泳]分析和蛋白质印迹分析。对表达产物行Ni2+-NTA亲和层析纯化、凝血酶酶切和高效液相色谱（HPLC）纯化后，行 SDS-Tricine-PAGE分析和飞行质谱分析，并以 ELISA 方法检测其免疫活性。 结果 PCR 扩增获得的条带与预期的DNA表达片段大小一致，重组质粒 pET-G 测序结果与预期完全一致。SDS- Tricine-PAGE 和蛋白质印迹分析显示 E.coli BL21（DE3）的表达产物相对分子质量与预期相符。经亲和层析纯化、凝血酶酶切和 HPLC 纯化后得到了完整的重组胰高血糖素-甘氨酸衍生物，SDS-Tricine-PAGE 分析显示其相对分子质量约为 3500，飞行质谱分析相对分子质量为 3531，二者基本一致。ELISA 检测表明重组胰高血糖素-甘氨酸衍生物具有胰高血糖素免疫活性。 结论 采用基因工程技术在大肠杆菌中成功表达了胰高血糖素-甘氨酸衍生物，为通过体外酰胺化途径研制酰胺化胰高血糖素奠定了基础。     

单链抗体与力达霉素融合蛋白在链霉菌中的表达

 目的 构建可表达力达霉素（LDM）辅基蛋白基因与抗 IV型胶原酶单链抗体（scFv）融合蛋白的重组菌株，获得既具有靶向性、又具有抗肿瘤活性的 scFV-LDM。方法 以大肠埃希菌-链霉菌穿梭质粒 pBS03 为基础构建同源双交换质粒 pBS-scFv，利用接合转移将该质粒转入球孢链霉菌（S. globisporus）C-1027，根据抗性差异筛选获得重组菌株。用 PCR、DNA 印迹法对重组菌株进行验证。用 SDS-PAGE 检测重组菌株中目的融合蛋白的表达。用抑菌圈试验检测重组菌株发酵液的抑菌活性。 结果 经同源重组得到重组菌株 S. globisporus C-1027- scFv。PCR 显示重组菌株扩增产物与预期一致，DNA 印迹分析显示重组菌株得到与预期吻合的杂交片段，表明 scFv-LDM 融合蛋白基因已经成功取代 LDM 辅基蛋白基因。SDS-PAGE 结果显示重组菌株不再产生辅基蛋白。抑菌圈试验显示重组菌株发酵液在第 5 天能够检测到抑菌活性。初步结果表明，重组菌株可产生 scFv-LDM 融合蛋白。 结论 成功构建了可产生 scFv-LDM 融合蛋白且具有分泌活性的重组菌株。这对改造和研发新型单抗导向药物有实际意义。     

PCV2-ORF2 去信号肽基因的克隆与原核表达及对小鼠免疫效力的评价

 目的 通过基因工程的方法表达猪圆环病毒 2 型的 ORF2 蛋白,并对表达蛋白进行小鼠免疫效力评价,为猪圆环病毒病疫苗的研制提供技术支持。 方法 根据 GenBank 中猪圆环病毒 2 型（PCV-2) ORF2 基因序列,设计了 1 对引物。从疑似断奶仔猪多系统衰竭综合症（PMWS）的死亡仔猪病料中提取基因组 DNA,用 PCR 方法扩增出 ORF2 去信号肽基因,将该基因克隆至原核表达载体 pET32a,获得重组质粒,经 PCR﹑酶切以及序列分析鉴定,表明插入的片段为目的基因,插入的位置、大小和阅读框均正确,成功构建了重组质粒 pet32a-ORF2,阳性重组质粒转化大肠杆菌 Blgold（DE3）,用 IPTG 诱导表达并确定表达的最佳条件,表达产物经纯化后,以每只小鼠 0.2 mg 免疫 5 周龄的雌性小鼠,然后用 ELISA 检测免疫小鼠血清中抗体的产生情况。 结果 纯化的重组蛋白能使小鼠产生了抗猪圆环病毒的特异性抗体,从第 7 d 开始产生抗体,第 28 d 抗体水平达到最高,抗体可维持 7 周以上,并具有良好的安全性。 结论 表达目的蛋白能在小鼠体内产生特异性抗体,为进一步研究猪圆环病毒亚单位疫苗奠定了基础。     

甘氨酸和 Triton X-100 对 ZZ-EGFP 融合蛋白分泌表达影响的初步研究

 目的 考察不同浓度的甘氨酸和 Triton X-100 对葡萄球菌蛋白质 A ZZ 亲和肽-增强型绿色荧光蛋白（ZZ-EGFP）融合蛋白在大肠杆菌分泌表达的影响。 方法 研究设计为两因素三水平析因设计。向液体培养基中分别加入终浓度 0、1%、2% 的甘氨酸和 0、1%、2% 的 Triton X-100（共 9 种组合方式，终浓度均为 0 者为对照组），诱导大肠杆菌周质腔内 ZZ-EGFP 融合蛋白泄漏到液体培养基中，以培养上清液荧光强度为观察指标，通过 ZZ- EGFP 融合蛋白浓度-荧光强度标准曲线快速检测培养基中 ZZ-EGFP 融合蛋白表达量。 结果 培养基中分别加入 1%、2% 甘氨酸或 1%、2% Triton X-100 培养后，培养上清液荧光强度分别为 283 ± 11、711 ± 19 和 622 ± 25、733 ± 25，与对照组（74 ± 5）比较，组间差异均有统计学意义（甘氨酸：F = 10.881，P = 0.024；Triton X-10：F = 12.848，P = 0.018）；而且甘氨酸与 Triton X-100两者之间有交互效应（F = 5.441，P = 0.005），其中培养基中同时含有终浓度 2% 甘氨酸及 1% Triton X-100 时培养上清液荧光强度最高（1854 ± 45），ZZ-EGFP 融合蛋白分泌表达量达到 10.4 mg/L，与对照组（0.94 mg/L）相比提高了 11 倍。 结论 甘氨酸和 Triton X-100 能提高 ZZ-EGFP 融合蛋白在液体培养基中的分泌表达量。     

人核糖体蛋白S15a在大肠癌组织中的表达及其意义

 目的 研究人核糖体蛋白S15a（RPS15a）在大肠癌组织中的表达情况及其与大肠癌发生发展的关系。方法 大肠癌及癌旁正常组织取自82例术前未经放疗、化疗患者的手术切除标本。利用RPS15a多克隆抗体进行免疫组织化学 SP染色，检测RPS15a在大肠癌及正常组织中的表达。结果 RPS15a阳性表达率在大肠癌组织中为72.0%（59/82），在癌旁正常组织中为40.2%（33/82），二者间差异有统计学意义（χ2 = 17.76，P < 0.05）；在浆膜浸润大肠癌组织中为80.8%（42/52），在无浆膜浸润大肠癌组织中为56.7%（17/30），二者间差异有统计学意义（χ2 = 5.47，P < 0.05）；在无淋巴结转移者为58.3%（21/36），在有淋巴结转移者为82.6%（38/46），二者间差异有统计学意义（χ2 = 5.89，P < 0.05）。结论 RPS15a在大肠癌组织中呈高表达状态，RPS15a基因可能在大肠癌的发生、发展中起重要作用。     

重组人 PTP1B 的原核高效表达及TFLC的体外抑制剂实验

 目的 高效诱导表达并纯化具有活性的重组 PTP1B 融合蛋白（rhPTP1B）,进行酶活性及抑制剂的实验。方法 重组体 pGEX-hPTP1B 转化 E.coliDH5α,诱导表达 rhPTP1B 并优化条件后,超声取细胞裂解上清液经 GST 亲和层析柱纯化后,利用 rhPTP1B 水解特异性底物 4-硝基苯基磷酸二钠盐（pNPP-Na2）的磷酸基团而产生颜色反应来测定 rhPTP1B 活性,并分析其与 rhPTP1B 浓度、底物浓度及反应温度的关系,钒酸钠（Na3VO4）抑制类型的研究,老鹰茶总黄酮（TFLC）对 rhPTP1B 的抑制性作用和浓度依赖关系。 结果 rhPTP1B 表达的最适条件是在 34 ℃ 和 2.0 mmol/L IPTG 下诱导表达 10 h,可被 GST 亲和层析法分离纯化;其活性随酶浓度及底物浓度的增加而增加,35 ℃ 时 rhPTP1B 的活性最大;钒酸钠的作用机制可能为非竞争性抑制作用;TFLC 对 rhPTP1B 有明显的抑制作用。结论 诱导表达纯化的 rhPTP1B 融合蛋白可用于抑制剂的研究,TFLC 对 rhPTP1B 的强抑制作用可能是其降低糖尿病大鼠血糖浓度的机制之一。     

IL-12双亚基共表达载体的构建及在人骨髓间充质干细胞中的表达

 目的 构建人 IL-12（hIL-12）p40 和 p35 双亚基真核共表达载体并转染人骨髓间充质干细胞（hMSC）。方法 根据 hIL-12 p40 和 p35 亚基全长 cDNA 序列分别设计合成引物行 PCR 扩增，将扩增所得 p40 和 p35 片段采用 overlap PCR 法拼接，获得的 rhIL-12 融合基因与 pGEM-T Easy 质粒连接，将鉴定正确的 pGEM-T/rhIL-12 重组质粒克隆至 pcDNA3.1（+）真核表达质粒中，构建 pcDNA3.1（+）/rhIL-12 真核表达载体，并进行测序鉴定。将鉴定正确的 pcDNA3.1（+）/rhIL-12 经脂质体介导转染 hMSC，同时以转染 pcDNA3.1（+）空质粒作为对照组，在倒置显微镜下观察细胞生长形态；在转染后第 4 天采用蛋白质印迹法检测细胞培养上清液中 rhIL-12 融合基因的表达；另分别在转染后第 2、4、6、8、10、12、14 天，采用 ELISA 方法检测细胞培养上清液中 rhIL-12 融合基因的表达水平。 结果 PCR 扩增结果显示特异性扩增出 p40（1000 bp）、p35（600 bp）、rhIL-12（1600 bp）片段，均与预期 DNA 表达片段大小一致。pcDNA3.1（+）/rhIL-12 测序显示克隆的 rhIL-12 基因序列与报告序列完全相同。倒置显微镜下观察，可见转染 pcDNA3.1（+）/rhIL-12 的 hMSC 生长形态和生长速度与对照组 hMSC 相比均无明显差异。蛋白质印迹和 ELISA 检测显示，转染 pcDNA3.1（+）/rhIL-12 的 hMSC 培养上清液中可见 rhIL-12 融合蛋白的持续表达；而对照组中均未检测到 rhIL-12 融合蛋白表达。 结论 成功构建了 hIL-12 p40 和 p35 双亚基真核共表达载体 pcDNA3.1（+）/rhIL-12，为利用 hIL-12 进行非病毒载体抗肿瘤基因治疗奠定了基础。     

可溶性HLA-G1与NK-92细胞表面ILT2及KIR2DL4受体相互作用的研究

 目的 探讨可溶性 HLA-G1（sHLA-G1）对人 NK-92 细胞杀伤活性的抑制与细胞表面免疫球蛋白样转录分子 2（ILT2）和杀伤细胞免疫球蛋白样受体 2DL4（KIR2DL4）受体的关系。 方法 ①通过原核表达技术获得 sHLA-G1 重组蛋白（重组蛋白），并采用蛋白质印迹法进行鉴定。②取 NK-92 细胞，加入终浓度 20 μg/ml 的重组蛋白分别培养 10、30 min，再分别加入抗 HLA-G1/G5、抗ILT2 和抗 KIR2DL4 抗体，采用流式细胞术检测各组 NK-92 细胞表面 sHLA-G1 和 ILT2、KIR2DL4 受体表达阳性率；以 NK-92 细胞单独培养作为对照组。③以人白血病 K562 细胞为靶细胞，以经不同方式处理的 NK-92 细胞为效应细胞，效靶比为5:1，共同培养 2 h，采用流式细胞术检测 NK-92 细胞对 K562 细胞的杀伤率。NK-92 细胞处理方式为单纯重组蛋白处理（分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）和表面受体封闭 + 重组蛋白处理（分别加入抗 ILT2、抗 KIR2DL4、抗 LT2 + 抗 KIR2DL4 抗体培养 30 min，再分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）。 结果 ①蛋白质印迹分析表明所获重组蛋白为带有组氨酸标签的特异蛋白。②NK-92 细胞与 20 μg/ml 重组蛋白共培养 30 min 后，sHLA-G1 表达阳性率明显高于而 ILT2、KIR2DL4 受体表达阳性率均明显低于对照组（均 P < 0.05）。③以终浓度 0、10、20 μg/ml 的重组蛋白处理的 NK-92 细胞对 K562 细胞的杀伤率分别为 39.79% ± 2.00%、27.79% ± 0.75%、21.36% ± 0.67%（两两比较，均 P < 0.01）；单独封闭 ILT2 受体，杀伤率分别为 23.09% ± 1.63%、21.13% ± 0.38%、18.42% ± 0.47%（两两比较，均 P < 0.01）；单独封闭 KIR2DL4 受体，杀伤率分别为 30.74% ± 0.44%、26.03% ± 0.38%、21.15% ± 0.35%（两两比较，均 P < 0.01）。 结论 sHLA-G1 通过与 NK-92 细胞表面 ILT2 和 KIR2DL4 受体直接结合而抑制 NK-92 细胞的杀伤活性。     

IL-5 receptor positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to inducegrowth and differentiation through the mIL-5 specific receptor(mIL-5R). The functional high-affinity mIL-5R is a heterodimercomposed of and ß chains. We investigated the expressionof mIL-5R and the responsiveness of B cells and eosinophilsto mIL-5 In X-linked immunodeficient (xld) mice. mIL-5R expressionanalyzed by using mAbs specific for and ß chainsrevealed that xld B cells had fewer mIL-5R⁺mIL-5Rß⁺than BALB/c B cells. In particular, a decrease in the numberof peritoneal mIL-5R⁺ B cells among Ly-1 B cells (known as B-1cells) was remarkable. Furthermore, the frequency of precursorsof mIL-5 responsive B cells in xld mice was 100-fold lower thanthat of BALB/c mice. Interestingly, sorted mIL-5R+ peritonealB cells from xld mice displayed a low response to mIL-5. Intraperltonealinjection of mIL-5 into BALB/c mice induced polyclonal IgM productionand an increase in the number of eosinophils. The same regimenfailed to induce an increase in the same parameters in xld mice.However, xld mice showed mIL-5-induced eoslnophilla in peripheralblood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injectedxld mice expressed both and ß chains of mIL-5, andresponded to mlL-5 with prolonged in vitro survival.     

Prediction of outcome and prognosis of patients on mechanical ventilation using body mass index, SOFA score, C-Reactive protein, and serum albumin

Context:

Body mass index (BMI), serum albumin, and C-reactive protein (CRP) appear to be major determinants of hospitalization.

Aim:

To determine the predictive ability of BMI, Sequential Organ Failure Assessment (SOFA score), serum albumin, and CRP to assess the duration and outcome of mechanical ventilation (MV).

Materials and Methods:

Thirty patients aged >18 years who required mechanical ventilation (MV) were enrolled for the study. They were divided into two groups; patients who improved (Group 1), patients who expired (Group 2). Group 1 was further divided into two groups: patients on MV for <5 days (Group A), and patients on MV for >5days (Group B). BMI and SOFA score were calculated, and serum albumin and CRP were estimated.

Results and Discussion:

Out of the 30 patients, 18 patients successfully improved after MV (Group 1) and 12 patients expired (Group 2). Among the 18 patients in group 1, ten patients improved within 5 days (Group A) and 8 patients after 5 days (Group B). SOFA score and CRP were significantly increased (P value 0.0003 and 0.0001, respectively) in group 2 when compared to group 1. CRP >24.2 mg/L or SOFA score >7 at the start of MV increases the probability of mortality by factor 13.08 or 3.92, respectively The above parameters did not show any statistical difference when group A was compared to group B.

Conclusion:

Simple, economic and easily accessible markers like CRP and assessment tools of critically ill patients with SOFA score are important determinants of possible outcomes of a patient from MV.     

Smoking,sperm quality and testosterone level

Dear Sir, Trummer et al. reported on the semen parameters of 1154 infertilemen. These authors divided their sample into smokers and non-smokers,and concluded that smoking does not affect conventional semenparameters (Trummer et al., 2002). However this study (likemost such studies) was cross-sectional. I want to suggest thathad the authors conducted a longitudinal study on normal fertilemen, they would have     

Relationship between protein concentrations in embryological fluids and maternal serum and yolk sacsize during human early pregnancy

Coelomic fluid (n = 57), amniotic fluid (n = 61) and maternalserum (n=81) were obtained from normal pregnancies between 7.7and 13.9 weeks and assayed for total protein, -fetoprotein (FP),albumin and pre-albumin. The mean concentration of total proteinin matched samples was 18 times higher in maternal serum thanin the coelomic fluid and 54 times higher in the coelomic fluidthan in amniotic fluid. The concentrations of total protein,albumin and pre-albumin decreased and that of FP increased inmaternal serum with advancing gestation. The yolk sac volumeand the concentrations of total protein in the coelomic andamniotic fluids increased with gestational age. No differencewas found for the crown–rump length, yolk sac volume andprotein concentration in the coelomic fluid between two groupspresenting with low and high maternal serum pre-albumin concentrations.Before 11 weeks gestation, significant correlation was onlyfound between yolk sac volume and coelomic fluid concentrationof pre-albumin as evaluated by both electrophoresis and immunonephelometry.These results suggest that during the first trimester of normalpregnancy, the placental metabolism and transfer rate of proteinsis not directly influenced by the concentrations of proteinin the maternal circulation and that the transfer of proteinsthrough the amniotic membrane is limited. These results alsoindicate that during that period the secondary yolk sac maycontribute to the protein content of the exocoelomic cavity,and that the embryo and its yolk sac and subsequently the fetusare the main source of the proteins present in the amnioticfluid.     

The influence of the site of sperm deposition and mode of oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates

The aims of this study were to examine, in a prospective, controlledway, the effect of the sperm deposition site in the oocyte andthe mode of oolemma breakage in intracytoplasmic sperm injection(ICSI) on fertilization and embryo development rates. In thefirst trial (100 cyclesin total), the spermatozoa were depositedfurther from themeiotic spindle (polar body at the 12 o'clockposition) in half of the oocytes (n = 649), while in the otherhalf(n = 605) the spermatozoa were deposited nearer to themeioticspindle (polar body at the 6 o'clock position). In the secondtrial (6860 oocytes in 624 cycles), five different modes ofmembrane breakage (the reaction of the oolemmato the penetratinginjection needle) at the moment of injection were noted: oolemmabreakage, type A pricking once, no suction (n = 1401); typeB, pricking once, smallsuction (n = 2761); type C, prickingonce, long suctionin = 2310); type D, pricking twice or more,no or small amount of suction (n = 259); and type E, prickingtwiceor more, long suction (n = 129). No differences were observedbetween the 12 and 6 o'clock positions in the survival rate(90 and 90% respectively) and in the normal fertilization rates(78 and 77% respectively). Significantly more transfer qualityembryos (50% fragmentation) were obtained in the 6 o'clock positiongroup (83%) than in the 12 o'clock position group (79%). Inthe second trial, significantly lower survival rates were notedafter membrane breakage type A (82%) than after breakages oftypes B, C, D and E (93, 92, 88 and 88% respectively). Therewere no significant differences present in the normal fertilizationrates (70, 72, 70, 71 and 73% for types A-E respectively), butsignificantly more freeze quality embryos (20% fragmentation)were obtained after injection B (65%) than after injection typesA, C, D and E (59, 61, 55 and 51%respectively). In conclusion,the site of sperm deposition inthe oocyte does not influencethe normal fertilization rate but does affect the embryo developmentrate. Furthermore, the mode of membrane breakage does not influencethe normal fertilization rate but does affect oocyte survivalandembryo development rates.     

Recombination hotspot associated factors specifically recognize novel target sequences at the site of interchromosomal rearrangements in T-ALL patients with t(8;14)(q24;q11) and t(1;14)(p32;q11)

A DNA binding protein was identified which binds to two noveltarget-like sequences: (I) at the 5' flanking site of the breakpointjunction of chromosome 8 in a patient with T-acute lymphoblastlcleukemla (ALL) carrying the t(8;14)(q24;q11) rearrangement and(II) on chromosome 1 in three of five T-ALL patients with thet(1;14)(p32;q11) rearrangement. This protein [provisionallycalled recombination hotspot associated factor (ReHF-1)] wasalso found to bind to a similar target sequence that is presentImmediately at the 3' end of the human V3 gene segment. In asmall number of lines tested, the ReHF-1 protein was expressedin lineage T cells and in a number of B cell precursor ALLswhose TCR locus has been rearranged. The molecular weight ofReHF-1 protein was determined to be 30 kDa by UV cross-linkinganalysis. Gel filtration chromatography and sedimentation velocitycentrifugation analyses indicate that the ReHF-1 protein existsas a multimeric protein in its native form. These data mightsuggest a possible role for this protein in the rearrangementof the TCR locus. Furthermore, another protein, ReHF-2, thatappears to have strict sequence specificity was found to bindonly to the complementary single strand of the target sequence.The interaction of these proteins with a conserved target sequenceat the chromosomal breakpoint junction might suggest that theyare involved In a novel enzymatic mechanism reminiscent of thegeneral features of DNA recombination or replication eventsIn Escherichia coli or Saccharomyces cenvisiae.