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基因修饰细胞治疗产品非临床研究与评价技术指导原则(试行)

基因修饰细胞治疗产品非临床研究与评价技术指导原则(试行)

.20212一、前言 (1)二、适用范围 (1)三、总体考虑 (1)四、受试物 (2)五、动物种属/模型选择 (3)六、概念验证 (4)七、药代动力学 (5)八、非临床安全性 (6)(一)总体安全性考虑 (6)(二)基因表达产物的风险评估 (6)(三)插入突变风险评估 (7)(四)载体动员(Vector mobilisation)和重组风险评估8九、对特定类型基因修饰细胞产品的特殊考虑 (8)(一)基因修饰的免疫细胞(CAR或TCR修饰的T细胞/NK细胞) (8)(二)诱导多能干细胞(iPS)来源的细胞产品 (10)(三)基因编辑的细胞产品 (10)一、前言近年来,随着基因修饰技术的迅速发展,基因修饰细胞治疗产品已成为医药领域的研究热点。

由于基因修饰细胞治疗产品物质组成和作用方式与一般的化学药品和生物制品有明显不同,传统的标准非临床研究策略和方法通常并不适用于基因修饰细胞治疗产品。

为规范和指导基因修饰细胞治疗产品非临床研究和评价,在《细胞制品研究与评价技术指导原则》基础上,根据目前对基因修饰细胞治疗产品的科学认识,制定了本指导原则,提出了对基因修饰细胞治疗产品非临床研究和评价的特殊考虑和要求。

随着技术的发展、认知程度的深入和相关研究数据的积累,本指导原则将不断完善和适时更新。

二、适用范围本指导原则适用于基因修饰细胞治疗产品。

基因修饰细胞治疗产品是指经过基因修饰(如调节、修复、替换、添加或删除等)以改变其生物学特性、拟用于治疗人类疾病的活细胞产品,如基因修饰的免疫细胞(如T细胞、NK细胞、树突状细胞和巨噬细胞等)和基因修饰的干细胞(如造血干细胞、多能诱导干细胞等)等。

三、总体考虑非临床研究是药物开发的重要环节之一。

对于基因修饰细胞治疗产品,充分的非临床研究是为了:1)阐明基因修饰的目的、功能以及产品的作用机制,明确其在拟定患者人群中使用的生物学合理性;2)为临床试验的给药途径、给药程序、给药剂量的选择提供支持性依据;3)根据潜在风险因素,阐明毒性反应特征,预测人体可能出现的不良反应,确定不良反应的临床监测指标,为制定临床风险控制措施提供参考依据。

RECSIT1.1中英文对照全文

RECSIT1.1中英文对照全文

New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)新版实体瘤疗效评价标准:修订的RECIST指南(1.1版本)Abstract摘要Background背景介绍Assessment of the change in tumour burden is an important feature of the clinical evaluation of cancer therapeutics: both tumour shrinkage (objective response) and disease progression are useful endpoints in clinical trials. Since RECIST was published in 2000, many investigators, cooperative groups, industry and government authorities have adopted these criteria in the assessment of treatment outcomes. However, a number of questions and issues have arisen which have led to the development of a revised RECIST guideline (version 1.1). Evidence for changes, summarised in separate papers in this special issue, has come from assessment of a large data warehouse (>6500 patients), simulation studies and literature reviews.临床上评价肿瘤治疗效果最重要的一点就是对肿瘤负荷变化的评估:瘤体皱缩(目标疗效)和病情恶化在临床试验中都是有意义的判断终点。

RNA 6000 Nano Kit for 2100 Bioanalyzer Systems Qui

RNA 6000 Nano Kit for 2100 Bioanalyzer Systems Qui

RNA 6000 Nano Kit for 2100 Bioanalyzer SystemsQuick GuideThe complete RNA 6000 Nano Kit for 2100 Bioanalyzer Systems Kit Guide can be found in the online help of the Agilent 2100 Expert software.Kit ComponentsAgilent RNA 6000 Nano Kit (5067-1511)Agilent RNA 6000 Nano Chips Agilent RNA 6000 Nano Reagents (5067-1512) & Supplies25 RNA Nano Chips (yellow) RNA 6000 NanoLadder (1 vial, 5067-1529)2 Electrode Cleaners (blue) RNA 6000 Nano Dye Concentrate (1 vial)(green) RNA 6000 Nano Marker (2 vials)Syringe Kit (red) RNA 6000 Nano Gel Matrix (2 vials)1Syringe 4 Spin Filters (5185-5990)Tubes for Gel-Dye Mix30 Safe-Lock Eppendorf Tubes PCR clean (DNase/RNase free) for gel-dye mixFor Research Use OnlyNot for use in Diagnostic Procedures.Assay PrinciplesAgilent RNA kits for the 2100 Bioanalyzer system contain chips and reagents designed for sizing and analysis of RNA fragments. Each chip contains an interconnected set of microchannels that is used for separation of nucleic acid fragments based on their size as they are driven through it electrophoretically. This kit is designed for use with the 2100 Bioanalyzer system only.Applications and KitsAgilent RNA kits are designed for the analysis of total RNA (eukaryotic, prokaryotic, and plant) and mRNA samples. Available kits: Agilent RNA 6000 Nano kit (5067-1511), RNA 6000 Pico kit (5067-1513) and Small RNA kit (5067-1548) Storage Conditions•Freeze unopened RNA ladder at -28–-15°C (-18–5°F). Prepared ladder aliquots need to be stored at -28–-15°C (-18–5°F). Keep all other reagents and reagent mixes refrigerated at 2–8°C (36–46°F) when not in use to avoid poor results caused by reagent decomposition.2•Protect dye and dye mixtures from light. Remove light covers only when pipetting. Dye decomposes when exposed to light.•Store the chips at room temperature.Equipment Supplied with the Agilent 2100 Bioanalyzer System•Chip priming station (5065-4401)•IKA vortex mixerAdditional Material Required (Not Supplied)•RNaseZAP® recommended for electrode decontamination and routine electrode cleaning •RNase-free water recommended for routine electrode cleaning•Pipettes (10µL and 1000µL) with compatible tips (RNase-free, no filter tips, no autoclaved tips)•0.5mL and 1.5mL microcentrifuge tubes (RNase-free)•Microcentrifuge ( 13000g)•Heating block or water bath for ladder/sample preparationSample preparationFor total RNA or mRNA analysis, the sample concentration must be within the specified range. If the concentration of your particular sample is above this range, dilute with RNase-free water.SpecificationsSetting up the Chip Priming Station1Replace the syringe:a Unscrew the old syringe from the lid of the chip priming station.b Release the old syringe from the clip. Discard the old syringe.c Remove the plastic cap of the new syringe and insert it into the clip.d Slide it into the hole of the luer lock adapter and screw it tightly to the chip priming station.2Adjust the base plate:a Open the chip priming station by pulling the latch.b Using a screwdriver, open the screw at the underside of the base plate.c Lift the base plate and insert it again in position C. Retighten the screw.Physical Specifications Analytical SpecificationsTotal RNA AssaymRNA Assay Analysis time30min Quantitative range 25–500ng/µL 25–250ng/µL Samples per chip 12Qualitative range 5–500ng/µL 5–250ng/µL Sample volume 1µL Sensitivity (S/N>3)5ng/µL in water25ng/µL in water Kit stability 4 months Quantitative precision (within a chip)10% CV 10% CV Kit size25 chips12 samples/chip = 300 samples/kitQuantitative accuracy11Determined analyzing the RNA ladder as sample20%20%Maximum salt concentration in sample100mM Tris 0.1mM EDTA or 125mM NaCl 15mM MgCl 2100mM Tris 0.1mM EDTA or 125mM NaCl 15mM MgCl 233Adjust the syringe clip:a Release the lever of the clip and slide it up to the top position.Essential Measurement Practices•Handle and store all reagents according to the instructions on the label of the individual box.•Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results.•Allow all reagents to equilibrate to room temperature for 30min before use. Thaw samples on ice.•Protect dye and dye mixtures from light. Remove light covers only when pipetting. The dye decomposes when exposed to light and this reduces the signal intensity.•Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results.•Always wear gloves when handling RNA and use RNase-free tips, microcentrifuge tubes and water.•It is recommended to heat denature all RNA samples and RNA ladder before use for 2min and 70°C (once) and keep them on ice.•Do not touch the 2100 Bioanalyzer instrument during analysis and never place it on a vibrating surface.•Always vortex the dye concentrate for 10s before preparing the gel-dye mix and spin down afterwards.•Use a new syringe and electrode cleaners with each new kit.•Use loaded chips within 5min after preparation. Reagents might evaporate, leading to poor results.•To prevent contamination (e.g. RNase), it is strongly recommended to use a dedicated electrode cartridge for RNA assays.•Perform the RNase decontamination procedure for the electrodes daily before running any assays. Refer to the kit guide for details on electrode cleaning and decontamination.Agilent RNA 6000 Nano Assay ProtocolPreparing the RNA Ladder1Spin the ladder down and pipette in an RNase-free vial.2Heat denature the ladder for 2min at 70°C.3Immediately cool the vial on ice.4Prepare aliquots in recommended 0.5mL RNase-free vials with the required amount for typical daily use.5Store aliquots at -28–-15°C (-18–5°F). After initial heat denaturation, the frozen aliquots should not require repeated heat denaturation.6Before use, thaw ladder aliquots on ice (avoid extensive warming).Preparing the Gel1Pipette 550µL of RNA gel matrix (red ) into a spin filter.2Centrifuge at 1500 g ±20% for 10min at room temperature.3Aliquot 65µL filtered gel into 0.5mL RNase-free microcentrifuge tubes. Use filtered gel within 4 weeks. Store at2–8°C (36–46°F).Handling ReagentsThe dye can cause eye irritation. Because the dye binds to nucleic acids, it should be treated as a potential mutagen.Kit components contain DMSO. DMSO is skin-permeable and can elevate the permeability of other substances through the skin.✓Follow the appropriate safety procedures and wear personal protective equipment including protective gloves and clothes as well as eye protection.✓Follow good laboratory practices when preparing and handling reagents and samples.✓Always use reagents with appropriate care.✓For more information, refer to the material safety data sheet (MSDS) on .Agilent Technologies Inc. 2001-2022Printed in Germany, Edition: 11/2022*G2938-90037*Part No: G2938-90037 Rev. E.00Document No: SD-UF0000031 Rev. E.00Allow the RNA dye concentrate (blue ) to equilibrate to room temperatureVortex RNA dye concentrate (blue ) for 10µL of RNA marker (green ) in all 12sample wells and in the well marked.to find information on your local Contact Visit the Agilent website. It offers useful information, support, and current developments about the products and /en/product/automated-electrophoresis/bioanalyzer-systems .Put a new RNA chip on the chip priming station.µL of gel-dye mix in the well marked .mL and then close the chip priming µL of gel-dye mix in the wells marked .µL of prepared ladder in well marked .Pipette 1µL of sample in each of the 12 sample wells. Pipette 1(green ) in each unused sample well.Put the chip horizontally in the IKA vortexer and vortex for 1 9 µl gel-dye 9 µl gel-dye1 µl ladder 1 µl sample5 µl marker。

果蝇体内 RNAi 文库使用指南说明书

果蝇体内 RNAi 文库使用指南说明书

果蝇体内RNAi文库使用Guideline for Using in vivo RNAi Libraries of Drosophila郭健1, 2,严冬1, *1昆虫发育与进化生物学重点实验室,分子植物科学卓越创新中心,植物生理生态研究所,中国科学院,上海;2中国科学院大学,北京*通讯作者邮箱:引用格式:郭健,严冬. (2019). 果蝇体内RNAi文库使用. Bio-101 e1010274. Doi: 10.21769/BioProtoc.1010274.How to cite: Guo, J. and Yan, D. (2019). Guideline for using in vivo RNAi libraries of Drosophila. Bio-101 e1010274. Doi: 10.21769/BioProtoc.10101274. (in Chinese)摘要:目前RNAi是在果蝇体内对基因进行功能分析最有力的工具之一。

该技术使用简单,只需要一步遗传杂交,即可实现组织和时间特异性的基因干扰。

全基因组RNAi文库的开发使得转基因RNAi品系几乎涵盖了果蝇基因组中的每一个基因,并且使得大规模体内RNAi筛选成为可能。

然而,目前共有四个果蝇中心可提供RNAi品系,每个中心的RNAi品系又基于不同的载体,因此为挑选和使用果蝇RNAi品系造成了一定难度。

本文对各个果蝇中心的RNAi文库进行介绍,并为RNAi品系的使用提供指南。

关键词:果蝇,RNAi,体内筛选,Gal4-UAS材料与试剂1. 提供RNAi品系的果蝇中心:Vienna Drosophila Resource Center (VDRC): https://stockcenter.vdrc.atTransgenic RNAi Project (TRiP): https:///fly-in-vivo-rnaiNational Institute of Genetics in Japan (NIG-FLY): https://shigen.nig.ac.jp/fly/ nigflyTsingHua Fly Center (THFC): 实验步骤一、RNAi文库介绍1. VDRC果蝇中心维也纳的VDRC果蝇中心由Barry Dickson开创,其RNAi文库有着最高的基因组覆盖率,包括针对12,671个(91%) 蛋白编码基因的26,957个转基因RNAi品系,由GD文库、KK文库、和shRNA文库组成。

大鼠载脂蛋白 B100(Apo-B100)酶联免疫分析试剂盒 说明书

大鼠载脂蛋白 B100(Apo-B100)酶联免疫分析试剂盒 说明书

大鼠载脂蛋白B100(Apo-B100)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用检测范围:7.8ng/ml-500ng/ml最低检测限:1.95ng/ml特异性:本试剂盒可同时检测天然或重组的大鼠Apo-B100,且与其他相关蛋白无交叉反应。

有效期:6个月预期应用:ELISA法定量测定大鼠血清、血浆、细胞培养上清或其它相关生物液体中Apo-B100含量。

说明1.试剂盒保存:-20℃(较长时间不用时);2-8℃(频繁使用时)。

2.浓洗涤液低温保存会有盐析出,稀释时可在水浴中加温助溶。

3.中、英文说明书可能会有不一致之处,请以英文说明书为准。

4.刚开启的酶联板孔中可能会含有少许水样物质,此为正常现象,不会对实验结果造成任何影响。

概述载脂蛋白B100(Apo-B100)是血清LDL中最主要的载脂蛋白。

它的主要功能是结合和转运脂质,参与脂类代谢,是肝脏合成和分泌富含甘油三酯的VLDL所必需的载脂蛋白。

Apo-B100是CHD和动脉粥样硬化的危险因子。

载脂蛋白B100主要在肝脏合成,是由4536个氨基酸组成的单链多肽,分子量512723,是迄今世界上已阐明一级结构的分子量最大的蛋白质,是乳糜微粒、LDL和VLDL的主要结构蛋白质,能够与LDL受体相结合,可转运脂类到肝外组织。

实验原理用纯化的抗体包被微孔板,制成固相载体,往包被抗Apo-B100抗体的微孔中依次加入标本或标准品、生物素化的抗Apo-B100抗体、HRP标记的亲和素,经过彻底洗涤后用底物TMB显色。

TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。

颜色的深浅和样品中的Apo-B100呈正相关。

用酶标仪在450nm波长下测定吸光度(OD值),计算样品浓度。

试剂盒组成及试剂配制1.酶联板(Assay plate):一块(96孔)。

2.标准品(Standard):2瓶(冻干品)。

3.样品稀释液(Sample Diluent):1×20ml/瓶。

qPCR内参

qPCR内参

qPCR内参植物样品类型稳定性好SAND(At2g28390)TIP41(At4g34270)未知功能基因(At4g26410)SAND(At2g28390)F-box(At5g15710)YLS8(At5g08290)组织器官APT1,EF1α,UBQ5发育时期,组织器官EF1α,UBQ5逆境18s rRNA,25s rRNA发育时期,品种,逆境(紫外伤害)18s rRNA,S-腺苷蛋氨酸脱羧酶基因GAPDH 热激蛋白hsp90基因未知功能基因(TC139056)生物胁迫(免疫病)18s rRNA,EF1α盐胁迫EF1α,CYP冷害胁迫EF1α,Ribosomal protein L 2Dan-J蛋白基因肿瘤蛋白基因CYP,GAPDH,TUA发育时期,组织器官CAC,TIP41发育时期组织器官和逆境胁迫(干旱,盐,高温,低温)、激素UBC18,ACT7组织器官和发育时期UBQ4,UBQ10环境胁迫SamDC 发育时期ACT2/7,TUA 组织器官,光周期,品种EF1b,CYP2ABC转运因子基因F-box家族基因胰岛素降解酶基因CDPK蛋白激酶基因棉花发育时期,组织器官Histone 3,UBQ,UBQ1组织器官GAPDH 品种间25s rRNA葡萄发育时期GAPDH,ACT,SAND,EF1αAt4g34270At4g33380发育时期发育时期,组织器官,营养缺乏(硫、磷碳源),非生物胁迫(激素,冷害,甘露醇,盐)逆境胁迫拟南芥大麦组织器官,高温胁迫水稻马铃薯番茄杨树发育时期,组织器官,逆境胁迫(创伤,激素,锈病)短柄草大豆甘蔗样品间UBQ11,TUA形成层At4g34270籼粳稻品种间eEF-1a,ACT1马铃薯生物胁迫(免疫病)和逆境胁迫(盐,干旱)EF1α物种名称可选内参植物基因(EF-1α)多聚泛素酶基因(UBQ)、肌动蛋白基因(ACT)、α微管蛋白基因、β微管蛋白基拟南芥T1、EF1α、e1F4α、TUB2、TUB6、TUB9、UBQ5、UBQ10、UBQ11;水稻CT11、TUB、25s rRNA、UBC、UBQ5、UBQ10、EF1α、e1F4α;籼粳稻a、Eif-4a、UBC、GAPDH、UBQ-5、TBL、ACT1;短柄草EF1α、GAPDH、SamDC、TUA6、UBC18、UBQ4、UBQ1;大豆TUA、TUB、ELF1α、UBC2、ELF1b、CYP2、G6PD、UBQ10;棉花、Histone3、UBQ7、Actin2、CYP、UBQ1、UBQ2;马铃薯s Rrna、EF1α、TUB、CYP、Ribosomal protein L 2、甘蔗ACT、TUB、GAPDH、25s rRNA;杨树A、ACT2、UBQ7、EF1b、TUB、ACT11、E1F4b、CYP、At4g33380大麦酸脱羧酶基因、GAPDH、热激蛋白hsp90基因、TC139056 番茄J蛋白基因、肿瘤蛋白基因、TUA、CYP、GAPDH葡萄浆果α、GAPDH、MDH、PP2A、SAND、TIP41、TUA、TUB、UBC、UBQ-L40、UBQ10杨树表达明显表达不明显PP2A 18s Rrna TUA18s Rrna TUAUBC)、α微管蛋白基因、β微管蛋白基因;。

小鼠试剂盒,小鼠白介素11(IL-11酶联免疫检测ELISA试剂盒使用说明书

小鼠试剂盒,小鼠白介素11(IL-11酶联免疫检测ELISA试剂盒使用说明书

小鼠试剂盒,小鼠白介素11(IL-11酶联免疫检测ELISA试剂盒使用说明书国内权威的科研试剂供应商,乔羽生物专业经营进口分装和原装、国产的Elisa试剂盒,品质保证,技术严格,无效果退款退货。

Elisa kit规格:48孔配置/96孔配置酶标试剂:3 ml×1瓶(48)/6 ml×1瓶(96)本试剂仅供研究使用标本:血清或血浆上海乔羽生物专业供应:使用目的:本试剂盒用于测定小鼠血清、血浆及相关液体样本白介素11(IL-11)含量。

试验原理:IL-11试剂盒是固相夹心法酶联免疫吸附实验(ELISA).已知IL-11浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。

先将IL-11和生物素标记的抗体同时温育。

洗涤后,加入亲和素标记过的HRP。

再经过温育和洗涤,去除未结合的酶结合物,然后加入底物A、B,和酶结合物同时作用。

产生颜色。

颜色的深浅和样品中IL-11的浓度呈比例关系。

试剂盒内容及其配制试剂盒成份(2-8℃保存)96孔配置48孔配置配制96/48小鼠份酶标板1块板(96T)半块板(48T)即用型塑料膜板盖1块半块即用型标准品:80ng/ml 1瓶(0.6ml)1瓶(0.3ml)按说明书进行稀稀空白对照1瓶(1.0ml)1瓶(0.5ml)即用型标准品稀释缓冲液1瓶(5ml)1瓶(2.5ml)即用型生物素标记的抗IL-11抗体1瓶(6ml)1瓶(3.0ml)即用型亲和链酶素-HRP 1瓶(10ml)1瓶(5.0ml)即用型洗涤缓冲液1瓶(20ml)1瓶(10ml)按说明书进行稀释底物A 1瓶(6.0ml)1瓶(3.0ml)即用型底物B 1瓶(6.0ml)1瓶(3.0ml)即用型终止液1瓶(6.0ml)1瓶(3.0ml)即用型标本稀释液1瓶(12ml)1瓶(6.0ml)即用型自备材料1.蒸馏水。

2.加样器:5ul、10ul、50ul、100ul、200ul、500ul、1000ul。

guide rna设计原则

guide rna设计原则

guide rna设计原则Guide RNA设计原则Guide RNA (gRNA)是一种在基因编辑技术中常用的工具,用于指导Cas9蛋白酶进行特定基因位点的剪切和修复。

在设计gRNA时,需要遵循一些原则,以确保其在基因编辑过程中的高效性和特异性。

本文将介绍一些常见的gRNA设计原则。

1. 序列选择选择合适的gRNA序列是设计过程中的第一步。

gRNA通常由20个核苷酸组成,其中包含一个20个碱基的目标序列,该序列与目标基因位点的DNA序列互补。

在选择目标序列时,应遵循以下原则:- 避免选择重复序列或与其他基因相似的序列,以确保特异性。

- 选择目标序列中没有多个连续的鸟嘌呤或胸腺嘧啶碱基,以避免gRNA自身形成二级结构。

2. PAM序列选择PAM(protospacer adjacent motif)是在目标序列的末端,与Cas9蛋白酶结合的序列。

在选择PAM序列时,应注意以下原则:- 常用的Cas9蛋白酶来自Streptococcus pyogenes,其PAM序列为NGG(N代表任意核苷酸,G代表鸟嘌呤碱基)。

因此,在设计gRNA时,应选择目标序列末端的PAM序列为NGG。

3. 避免剪切位点过于靠近起始密码子在选择目标序列时,应避免选择靠近起始密码子的区域作为剪切位点,以避免对目标基因的翻译产生严重影响。

4. 避免剪切位点过于靠近外显子-内含子交界处在选择目标序列时,还应避免选择靠近外显子-内含子交界处的区域作为剪切位点,以避免对剪切后的修复过程产生不利影响。

5. 评估离靶位点为了确保gRNA的特异性,需要评估离靶位点的可能性。

离靶位点是指与目标序列相似的、但不完全互补的DNA序列。

为了减少离靶效应,应遵循以下原则:- 使用计算工具评估gRNA在整个基因组中的离靶位点数量。

- 避免选择与目标序列具有高度相似性的序列,尤其是在PAM序列的邻近位置。

6. 评估剪切效率和特异性设计好的gRNA需要进行实验验证,评估其剪切效率和特异性。

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AP® Biology2011 Scoring GuidelinesThe College BoardThe College Board is a not-for-profit membership association whose mission is to connect students to college success and opportunity. Founded in 1900, the College Board is composed of more than 5,700 schools, colleges, universities and other educational organizations. Each year, the College Board serves seven million students and their parents, 23,000 high schools, and 3,800 colleges through major programs and services in college readiness, college admission, guidance, assessment, financial aid and enrollment. Among its widely recognized programs are the SAT®, the PSAT/NMSQT®, the Advanced Placement Program® (AP®), SpringBoard® and ACCUPLACER®. The College Board is committed to the principles of excellence and equity, and that commitment is embodied in all of its programs, services, activities and concerns.© 2011 The College Board. College Board, ACCUPLACER, Advanced Placement Program, AP, AP Central, SAT, SpringBoard and the acorn logo are registered trademarks of the College Board. Admitted Class Evaluation Service is a trademark owned by the College Board. PSAT/NMSQT is a registered trademark of the College Board and National Merit Scholarship Corporation. All other products and services may be trademarks of their respective owners. Permission to use copyrighted College Board materials may be requested online at: /inquiry/cbpermit.html.Visit the College Board on the Web: .AP Central is the official online home for the AP Program: .Question 1During an investigation of a freshwater lake, an AP Biology student discovers a previously unknown microscopic organism. Further study shows that the unicellular organism is eukaryotic.(a)Identify FOUR organelles that should be present in the eukaryotic organism and describe thefunction of each organelle.(5 points maximum)(b)Prokaryotic cells lack membrane-bound organelles found in eukaryotes. However, prokaryotesmust perform many of the same functions as eukaryotes. For THREE of the organelles identified in part (a), explain how prokaryotic cells carry out the associated functions.(3 points maximum)Question 1 (continued)(c)According to the endosymbiotic theory, some organelles are believed to have evolved through asymbiotic relationship between eukaryotic and prokaryotic cells. Describe THREE observations that support the endosymbiotic theory.(4 points maximum)Describe three observations(1 point each)•Mitochondria contain their own DNA.•Chloroplasts contain their own DNA.•Mitochondria can self-replicate.•Chloroplasts can self-replicate.•Mitochondrial chromosomes are circular.•Chloroplast chromosomes are circular.•Mitochondrial chromosomes lack histones.•Chloroplast chromosomes lack histones.•Mitochondria contain ribosomes that are similar to bacterial ribosomes.•Chloroplasts contain ribosomes that are similar to bacterial ribosomes.•Inner membrane of mitochondria is similar the membrane of prokaryotes.•Inner membrane of chloroplasts is similar the membrane of prokaryotes.•Mitochondria can perform transcription and translation.•Chloroplasts can perform transcription and translation.•First amino acid in the polypeptides in mitochondria is fMet (N-formylmethionine).•First amino acid in the polypeptides in chloroplasts is fMet (N-formylmethionine).•Mitochondria are approximately the size of bacteria.•Chloroplasts are approximately the size of bacteria.•Mitochondria use many prokaryote-like enzymes.•Chloroplasts use many prokaryote-like enzymes.•Many antibiotics (e.g., rifampicin) interfere specifically with mitochondrial protein synthesis.General description of endosymbiotic theory (1 point)•Prokaryotic cells have been engulfed by and are living within ancestral/precursor eukaryotes.Question 2Organisms utilize a diversity of methods to obtain proper nutrition.(a)Some organisms digest food intracellularly, while others digest food extracellularly.(4 points maximum)•Identify ONE nonvertebrate organism that digests food intracellularly and describe the process.•Identify ONE nonvertebrate organism that digests food extracellularly and describe the process.(b)Describe TWO structural features of the human stomach and/or small intestine. For each, explainhow the structure relates to the function.(4 points maximum)Question 2 (continued)(c)Plants have a variety of mechanisms for obtaining nutrients. Describe TWO plant structures andexplain how each structure is utilized in nutrient uptake.(4 points maximum)Question 3Reproduction can be either asexual or sexual.Note: Points must be earned from parts (a), (b), and (c) in order to earn a maximum score of 10.(a)Using a specific example, describe how organisms can reproduce asexually.(3 points maximum)Discuss TWO evolutionary advantages of asexual reproduction.(2 points)•It is successful at low population density.•It eliminates the energy cost of finding a mate.•It exploits stable environments.•It is rapid and efficient.•It eliminates the energy cost of fertilization/pollination.•It eliminates the need for pollinators in plants.Question 3 (continued)(b)Identify THREE ways that sexual reproduction increases genetic variability. For each, explainhow it increases genetic diversity among the offspring.(6 points maximum)(c)Discuss TWO prezygotic isolating mechanisms that prevent hybridization between two species.Include in your discussion an example of each mechanism.(4 points maximum)Question 4The regulation of transpiration is an important homeostatic mechanism in plants. (a) Under controlled conditions, a transpiration experiment was conducted using two plant species.The data collected are shown in the figure below. Using the data from the experiment, calculate the rate of transpiration for species A and species B between the times of 5 and 15 minutes (show your work). Summarize the difference between the two transpiration rates. (3 points maximum) WATER LOSS VERSUS TIME FOR TWO PLANT SPECIES• Calculate transpiration rates, with units (1 point each; 2 points maximum).• Correct setups with incorrect results (1 point maximum).Species A (1 point) 3.6 mL H 2O - 1.2 mL H 2O 15 minutes - 5 minutes= 0.24 mL H 2O/100g/min (±0.02) OR 3.6 - 1.2 15 - 5 = 0.24 mL H 2O/100g/min (±0.02) OR equivalentQuestion 4 (continued) Species B (1 point) 1.8 mL H 2O - 0.4 mL H 2O 15 minutes - 5 minutes= 0.14 mL H 2O/100g/min (±0.02) OR 1.8 - 0.4 15 - 5 = 0.14 mL H 2O/100g/min (±0.02) OR equivalentSummarize the difference between the rates (1 point). • Species A is losing water or transpiring faster than species B.(b) Identify and explain THREE different structural or physiological adaptations that could account for the different transpiration rates of species A and B. (6 points maximum)Question 4 (continued)(c) Water potential (Ψ) is described by the following formulas.Ψ = Ψp + ΨsΨ = -iCRTDiscuss the variables in both formulas and how they affect water potential.(4 points maximum)•Discussion stating that the formula allows osmotic potential or water movement to be calculated or predicted (1 point).。

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