请各位大侠将下列凑乱的字母拼成单词，并告诉新单词和它的意思。_百度知道
请各位大侠将下列凑乱的字母拼成单词，并告诉新单词和它的意思。
a,p23.u,r17,r,s38,e,o,u24,l.i,r,z,y,o,g,o.p,l13.i,b19,e.a,r.p,c,s35,s.p.o,e,e,a,c22.a,h,r3,t,l,e,e21,g6,e,w,o,l,l,l,n30,r,e,y12,b,l,s,j,k36,p,a,a,r,e.s,a.f,i.n,l,p,a.h,e5,e,j.f,p,t7.i,e.i,s.p,c14,s37,u,r.r,t.o,k,p,r,h,y,h,n.o,e,r,p,t,o11,h,r,u,b,i,l,r,e.u,d,e,b,e,i,w,l28,r,h,t32.b,s4,y39,o.e,r.e,y,s29.t,c20.e,o,r.o,s,t,n.o,c,m,e,h,e31,d25,u.w,x,u,s,t,a,i33,l,m,t16.s,k10,b8,o27,n.u,r.p,a,b40,t34.c,e,r15,e,u,n,h,o,u1,n.l.s,a,r,u,t麻烦解决一下,t,t.o,z,l,n26,h,o.a,n,a,a,a9,b2,t，虽然题目有点多,t,s18.i,r,i,r.a
提问者采纳
puppet 木偶23.u,r
super 非常17,c,c,e,r,u
puzzle 谜,r.i,r,z,n.a,g,r.p,l
lion 狮子13.i,b
19,e.a,a.p,u,o,s.p.o,e,e,a,c
excuse 借口22,d
doll 玩偶25.o,r
ruler 尺子3.s,a,h,e
tape 磁带21,g
right 对的6,t,r,o,b,l,l,y,r,e,y
your 你的12,b,l,h,a,j,l,a,a,r,e,t
short 短的34,r,s
sweater 毛线衫 37,i,y
worry 担心39.o,p.e,i
skirt 裙子33,e
user 用户5,l,a,s
jeans 牛仔裤29,p,t
rabbit 兔子7.i,e.i,s,b
nice 美好的14,a,u,s
scarf 围巾38.r,t.o,r,p,r,h,h,h,n,n
funny 可笑的30,e,r,p,e,o
too 也 11,h,r.f,b,i,o,r,e.u,d.c,l.w,i,s,m,r.e,y.b,s
this 这4,w.p,e
whose 谁的31,e.e,y,n.t,c
small 小的28,e,t,n
balloon 气球26.h,e,u，拼图24,r,m,e,h,w,l,u,k
jacket 外套36,x,u.a,t,a,k,s,l,t
that 那16.s,k
like 喜欢10,b
bear 熊8,l,j.u,r.p,a,p,o,s
shoes 鞋子35,e,r
tiger 虎15,e,u,n.s,o,u1,t,o,o,t,t
they 他们32.f,e,r,i.n,a.o,s,n,a
panda 熊猫9,t
photo 照片不好意思,l.i,z,o.o.a,n,t,t,h,b
rubber 橡皮2.l,o
colour 颜色27,s
perhaps 也许18
提问者评价
其他类似问题
为您推荐：
其他2条回答
正确的;右边
喜欢;象;可能
你的, 你们的
十分的;非常
17.perhaps 或许, 多半
脑袋, 头顶
原谅; 致歉
傀儡, 木偶
小的, 少的
有趣的, 好笑的
他们, 它们
36.sweater 厚运动衫, ...
太多了....分开来发贴吧
字母的相关知识
等待您来回答
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23:14:00 | 来自
H6C和传奇GS4几乎前后脚上市
后者现在提车作业都好几个了，前者连个晒订单的都没。说明了什么？？？
引用 世界的最后一秒
23:14:00 发表于 主楼 的内容：
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说明长城的产能还没准备好，所以现在只是可以下订单，到货要2-3个月
引用 toto_lxn
23:15:19 发表于 1楼 的内容：
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23:27:01 | 来自
有晒单的…就是提车很慢。GS4现车主已经快成一个师了…
引用 夏天不懂春天的爱
23:27:01 发表于 2楼 的内容：
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23:30:52 | 来自
23:27:01 发表在 有晒单的…就是提车很慢。GS4现车主已经快成一个师了…我觉得GS4对H6的销量会冲击很大，看五月H6还能上3万不？
引用 世界的最后一秒
23:30:52 发表于 3楼 的内容：
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23:35:11 | 来自
说明6C该降价了吧！！！
批发代购章丘大葱、鲍芹、烤肉等名优特产
引用 康逸随心
23:35:11 发表于 4楼 的内容：
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23:53:55 | 来自
23:30:52 发表在 我觉得GS4对H6的销量会冲击很大，看五月H6还能上3万不？GS4内饰和后背箱是一大诟病…哎。
引用 夏天不懂春天的爱
23:53:55 发表于 5楼 的内容：
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00:04:47 | 来自
国产SUV市场就这么大，多一个神车肯定所有的车销量都会降，没啥奇怪的，坐等H6送购置税无息贷终身免保活动，大众都能领先降价，国产领头羊也可以玩啊，让后来的各种神车直接跟进玩烧钱，然后各种配置缩水，各种出问题，各种互黑，死几个就消停了，活到最后的就是英雄好汉
引用 Now2future
00:04:47 发表于 6楼 的内容：
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00:10:55 | 来自
GS4的做工堪比永源汽车！
引用 zoukun-09 00:10:55 发表于 7楼 的内容：
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00:12:43 | 来自
最好合资继续下探，合资紧凑SUV低配杀进12到15万区间，把所有国产紧凑SUV高配都划拉出良心企业行列，那论坛口水的场面，美得不敢想象，这工作就交给现代起亚和两田了。
引用 Now2future
00:12:43 发表于 8楼 的内容：
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00:19:08 | 来自
H6要降价现在最合适，现在冒出来的那么多国产SUV都是刚完成研发，进入批产阶段，该花的钱都花了，想赚的钱还没到手，这叫半渡而击之，效果最好。
引用 Now2future
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02:34:12 | 来自
长城一贯的拖沓…
引用 霸气y露
02:34:12 发表于 10楼 的内容：
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上传购车发票，点评购车服务感受，通过工作人员审核，特授予【真实报价员】专属勋章。
发表500字以上推荐口碑，点评客观公正，通过工作人员审核，特授予【推荐口碑】专属勋章。
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23:14:00 发表在
后者现在提车作业都好几个了，前者连个晒订单的都没。说明了什么？？？
说明你没有真正关注过h6 coupe，如果你真正关注过h6 coupe，你就应该看到过长城关于订车提车时间的告示。
引用 就爱看你装逼
02:40:09 发表于 11楼 的内容：
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08:35:17 | 来自
说的好，话粗理不粗。
引用 顶天立地-09 08:35:17 发表于 12楼 的内容：
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09:28:13 | 来自
02:40:09 发表在
说明你没有真正关注过h6 coupe，如果你真正关注过h6 coupe，你就应该看到过长城关于订车提车时间的告示。我当然不关注哈佛了，烂车一个还那么贵，我关注锐腾
引用 世界的最后一秒
09:28:13 发表于 13楼 的内容：
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所属：爱车：
完成汽车之家·知道升级任务，解答问答，并被提问者采纳为满意回答，可得解答达人一级勋章
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上传购车发票，点评购车服务感受，通过工作人员审核，特授予【真实报价员】专属勋章。
09:35:38 | 来自
23:35:11 发表在 说明6C该降价了吧！！！借你吉言…
引用 darkdj
09:35:38 发表于 14楼 的内容：
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参与汽车之家10周年“真交情，十年如初”活动，获此勋章，感谢一路上有你的陪伴。
09:45:21 | 来自
引用 秋物S
09:45:21 发表于 15楼 的内容：
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11:13:04 | 来自
对，现在h6降万八千最好
11:13:04 发表于 16楼 的内容：
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所属：爱车：
完成汽车之家·知道升级任务，解答问答，并被提问者采纳为满意回答，可得解答达人一级勋章
发表1500字以上满级口碑，观点独到，篇幅惊人，通过工作人员审核，特授予【满级口碑】专属勋章。
参加#2016法国欧洲杯专题#活动，赢取限量版勋章，精彩纷呈，尽在汽车之家。
上传购车发票，点评购车服务感受，通过工作人员审核，特授予【真实报价员】专属勋章。
分享购车价格，点评购车服务感受，通过工作人员审核，特授予【家de报价员】专属勋章。
00:19:08 发表在
H6要降价现在最合适，现在冒出来的那么多国产SUV都是刚完成研发，进入批产阶段，该花的钱都花了，想赚的钱还没到手，这叫半渡而击之，效果最好。
你斯，用计何其毒也
引用 15-05-09 11:23:54 发表于 17楼 的内容：
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完成汽车之家·知道升级任务，解答问答，并被提问者采纳为满意回答，可得解答达人一级勋章
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12:03:25 | 来自
11:23:54 发表在
你斯，用计何其毒也必须的，商场如战场么，就怕长城产能跟不上，无车可卖，目前市场就是，大家都卖大家都火，如果拼持刀，那必须有产能。
引用 Now2future
12:03:25 发表于 18楼 的内容：
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完成汽车之家·知道升级任务，解答问答，并被提问者采纳为满意回答，可得解答达人一级勋章
上传购车发票，点评购车服务感受，通过工作人员审核，特授予【真实报价员】专属勋章。
发表500字以上推荐口碑，点评客观公正，通过工作人员审核，特授予【推荐口碑】专属勋章。
参与汽车之家10周年“真交情，十年如初”活动，获此勋章，感谢一路上有你的陪伴。
参与汽车之家质量评价调研，反馈真实质量情况，特授予汽车之家“质量评价员”称号，并奖励专属勋章。
09:28:13 发表在 我当然不关注哈佛了，烂车一个还那么贵，我关注锐腾嘴上说不要，身体很诚实啊。。。
引用 就爱看你装逼
12:30:09 发表于 19楼 的内容：
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汽车之家公益系列活动，让我们一起抵制安全带卡扣！汽车之家呼吁大家一起来响应抵制危害安全用品的公益
2012年汽车之家问答问题数达5万的纪念活动，感谢您信赖汽车之家问答，在活动期间提出汽车生活中的问题
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畅聊2013年上海车展即将或者可能上市的新车，说出你的看法。
16:49:33 | 来自
23:53:55 发表在 GS4内饰和后背箱是一大诟病…哎。其实最大诟病应该是那1.3排量吧，虽然也是T，基础排量在那放着，平路真没什么问题，只是夏天要是开着空调地库还可以爬上去吗。。
开着比亚迪去给媳妇抓羊吃
引用 开着比亚迪的灰太狼
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在传奇GS4和启辰T70之间纠结，到底选哪个呢[待解决]
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如题，给位大大们能给点建议吗？这两款车价格都差不多，但是配置有很大区别，真的不知道怎么选择了！
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太平洋舰队新兵-1级
贡献(馒头)43, 距离下一级还需7贡献(馒头)
要看自己的实际使用要求
太平洋舰队下士-4级
贡献(馒头)246, 距离下一级还需54贡献(馒头)
肯定是小4了。
太平洋舰队新兵-1级
贡献(馒头)37, 距离下一级还需13贡献(馒头)
一句话，一分钱一分货，纠结没用。
太平洋舰队新兵-1级
贡献(馒头)38, 距离下一级还需12贡献(馒头)
一定小四呀！
太平洋舰队新兵-1级
贡献(馒头)18, 距离下一级还需32贡献(馒头)
当然GS4，T70是板车悬挂，而且屁股上的日字。。。。。。。
太平洋舰队列兵-2级
贡献(馒头)52, 距离下一级还需48贡献(馒头)
不要买T70&破日本车
太平洋舰队新兵-1级
贡献(馒头)30, 距离下一级还需20贡献(馒头)
?????的，肯定是?注小四的
太平洋舰队新兵-1级
贡献(馒头)30, 距离下一级还需20贡献(馒头)
选GS4的手动就好，GS4的自动是双离合不能选。T70是已经淘汰了的东西了。
太平洋舰队下士-4级
贡献(馒头)246, 距离下一级还需54贡献(馒头)
肯定是小4了！
太平洋舰队新兵-1级
贡献(馒头)26, 距离下一级还需24贡献(馒头)
哈哈！4是广汽出的，不是日本车？这？
太平洋舰队新兵-1级
贡献(馒头)16, 距离下一级还需34贡献(馒头)
你智商这么低？居然说广汽是日本车
太平洋舰队新兵-1级
贡献(馒头)29, 距离下一级还需21贡献(馒头)
你确定T70是板车？
太平洋舰队新兵-1级
贡献(馒头)29, 距离下一级还需21贡献(馒头)
两个车我都看过，如果是家用而且选自动的绝对是T70的好，但你要接受没有esp，如果手动你就选GS4，2.0实打实的动力跟1.3T开起来的感觉不一样，而且日产的发动机非常安静
太平洋舰队新兵-1级
贡献(馒头)26, 距离下一级还需24贡献(馒头)
你智商之高让我惊叹！我是专门给广汽供应继电器的厂家，每年的广汽的零件招标会我都会陪领导参加，你知道广汽采购的零件是哪些厂家生产的？/96的零件有日资背景，你知道？汽
太平洋舰队新兵-1级
贡献(馒头)19, 距离下一级还需31贡献(馒头)
t70其实不是逍客的配件，你看独立后悬挂比逍客少2个平衡杆，而且是双成冲压件，没有ESP，小日本车最好别出事故不然
太平洋舰队新兵-1级
贡献(馒头)19, 距离下一级还需31贡献(馒头)
T70，老逍客，稳定可靠，小毛病少，价格公道，性价比高
太平洋舰队新兵-1级
贡献(馒头)16, 距离下一级还需34贡献(馒头)
太平洋舰队新兵-1级
贡献(馒头)22, 距离下一级还需28贡献(馒头)
感谢兄弟们的建言&&我去试车了再说
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【求助】IVS4nt-14G→A这个突变是什么意思呢？
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IVS是内含子，4nt-14是什么意思就不清楚了。谢谢大家
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The question is quite complex. The mutation is a kind of splicing junction nuclrotide variation or conversion, the cited splicing junction mutation IVS4nt-14G-A is in intron 4 on minus strand with G at junction converts to A.According to Glossary at NCBI web site, the definition of splicing site or junction mutation is:& A mutation that alters or abolishes the specific sequence denoting the site at which the splicing of an intron takes place. Such mutations result in one or more introns remaining in the mature messenger RNA and can disrupt the generation of the protein product&.I am person who is very poor about molecular biology of mRNA splicing and prediction of results of splicing junction mutation. To predict the results of the mutation cited, you may need to download attached files: Information analysis of human splice site mutationsPeter K. Rogan, Brian M. Faux,*andThomas D. Schneiderversion = 1.73 of rfs.tex 1999 August 12 Abbreviated Title:Information at human splice site mutationsHuman Mutation 12: 153-171 (1998)ABSTRACTSplice site nucleotide substitutionscan be analyzed by comparing the individual informationcontents (Ri, bits)of the normal and variant splice junction sequences[].In the present study, we relatedsplicing abnormalitiestochanges in Ri values of 111 previouslyreported splice site substitutions in 41 different genes.Mutant donor and acceptor sites have significantly lessinformation than their normal counterparts.With one possible exception, primarymutant sites with & 2.4 bits were not spliced.Sites with Ri values bitsbut less than the corresponding natural site usuallydecreased but did not abolishsplicing.Substitutions thatproduced small changes in Riprobably do not impair splicing and are often polymorphisms.The Ri values of activatedcryptic sites were generally comparable toor greater than those of the corresponding natural rmation analysis revealedpre-existingcryptic splice junctions that are used instead of the mutatednatural site.Other cryptic sites were created or strengthened by sequencechanges that simultaneously altered the parison between normal and mutantsplice site Ri values distinguishes substitutions thatimpair splicing from those which do not,distinguishes null alleles from thosethat are partially functional,and detects activated cryptic splice sites.keywords:information theory,mRNA splicing,donor,acceptor,cryptic,mutation,polymorphism,walkerINTRODUCTIONMutations at splice sites make a significant contribution to human geneticdisease, since approximately 15% of disease-causing point mutations affectpre-mRNA splicing [].Mutations in splice sitesdecrease recognition of theadjacent exon and consequently inhibitsplicing of the adjacent intron[,].Splice site mutations mayresult in exon skipping, activation of cryptic splice sites, creationof a pseudo-exon within an intron, or intron retention[]:(1) Exonskipping, the most frequent outcome, is thought to result from failure of thenormal and mutant splice sites to define an exon. (2) Most cryptic mutationsactivate splice sites of the same typeand are typically locatedwithin a few hundred nucleotides ofthe natural site. This distance is probably limited by restrictions onthe length of theresultant exon [,].(3) Occasionally, mutationsthat are further awayfrom the natural splice site create cryptic sites that are activated inthe presenceof a nearby cryptic splice site of opposite polarity, producing a novelnon-coding exon within the intron.(4) Splice site mutations in very short or terminal intronscan result in intron retention [].In these instances,additional sequence elements may be requiredfor normal splicing[,,].Essential elements in donor and acceptor splicejunctions have been defined by consensus sequences [], byanalysis of nucleotide frequencies at each position in a splice site[] and by neural network prediction[].Each of these methods have limitations.Although the GT and AG positions adjacent to donor and acceptorsplice junctions are highly conserved,other positionsare more variable [,].The consensus sequence approximatesthe nucleotide frequencies at each position,and so it excludes thecontributions of less frequent nucleotides present ina proportion of natural splice sites.Splice site sequences that deviate from the consensusdo not necessarily produce significantly loweramounts of spliced mRNA [].Training a neural network requiressequences of both binding sites and sequences that are not bound[,].Generally, non-bound sequences are taken to be those remainingafter binding sites have been identified.However, these sequences docontain functional sites [,],so neural networksmay be inappropriatelytrained on overlapping data sets.In contrast,information-theory based modelsof donor and acceptor splice sites require only functional sitesand show whichnucleotides are permissible at both highly-conserved and variablepositions of thesesites [].Information is the only measure of sequence conservation which is additive[].The information content(Ri, in bits) of amember of a sequence family describes the degree to which that membercontributes to the conservation of the entire family[,].Ri is thedot product of a weight matrix derived from the nucleotide frequencies ateach position of a splice sitesequence database and thevector of a particular sequence.Individual informationis related to thermodynamic entropyand therefore to the free energy of binding[,].Sincesplice sites are recognized prior to intron excision [],the sequence of the splice site dictates the strength of thespliceosome-splice junctioninteractionand thus splice site use.It is our thesis thatthe strength of this interactionis related to the information content of the splice junction.A group of sites with similar sequence and function can be described andquantified by their corresponding distribution of individual informationcontents. The mean of this distribution of Ri values isbits for the 10 nucleotide long splice donor sites andbits for the 28 nucleotide long acceptor sequences[,],representing the average amount ofinformation required for splicing,Rsequence[,,].Strong splice sites have Ri values;weak sites have Ri values.Non-functional sites have Rivalues less than or equal to zero [,].Since mutations at splice sites lessen or abolish splicing at those sites,we investigated whether the Ri values of mutant splicesites were relatedto defects in mRNA processing and whethermutant, cryptic and the corresponding natural splice sites could be orderedbased on their respective Ri values.MATERIALS AND METHODSIndividual information analysisInformation content is defined as the number of choices needed to describe asequence pattern,using a logarithmic scale in bits[,].A set of either donor or acceptor splice junction recognition sites arealigned and the frequencies of bases at each position are determined.The weight matrix used to model the splice junctions is computed from (1)where f(b,l) is the frequency of each base b at position l inthe aligned binding site sequencesande(n(l)) is a sample size correction factor[]for the n sequences at position l used to create f(b,l)[].Thematrix,Riw(b,l), is a 2 dimensional array in which row b corresponds toone of the 4 nucleotides in DNA and column l is the position along thealigned set of splice junction recognition sites.
This individualinformation matrix represents the sequence conservation of eachnucleotide, measuredin bits of information.Riw(b,l) can be used to rank-order the sites, tosearch for new sites, to compare sites with one another, to compare sites toother quantitative data such as DNA-protein binding strength, and to detecterrors in databases [,].The individual information of a sequence j is the dot product between thesequence and the weight matrix: (2)where s(b,l,j) is abinary matrix for the jth sequence,in which cells have a value of 1 forbase b at position l and a value of 0 elsewhere.The mean of the distribution of Ri values of natural sites isRsequence[,]. The distribution of Ri values isapproximately Gaussian, however the lower and upper bounds are zero bits andthe Ri value of the consensus sequence.The null Ri distribution was determined by creating a random10,000 nucleotide sequencewith a Markov chain process that maintained the same mono- and dinucleotidecomposition as the human splice junction database[].The means of the splice donor and acceptor null distributionswere respectivelyandbits.The probability of observing eithera donor oracceptor sitewith Ri& 0in this random sequencewas 0.02 (Z = 2.0).The effects of nucleotide substitutionscan be evaluated by comparing the individualinformationof the common and variant alleles.The minimum fold change in binding affinity of twosites is,whereis the difference betweentheir respective individual information contents [].Computational toolshave been developedto investigate and display individual information.TheRiw(b,l)matrices were first computed from a set of 1799 splice donor and 1744 acceptorsequences [].To scan for potential sites orto determine the effects of a sequence changeon the normal and neighboringsites,
the individual information content of the donor or acceptor motif iscomputed for every site-length window in the sequence.To assess the effects of various substitutions on a specificdonor or acceptor site,Ri was computed for the normal and variant siteswith the programScanand displayed withMakeWalker, DNAPlot and Lister(Schneider.http://schneider.ncifcrf.gov/toms/walker).The Scan program uses theRiw(b,l) matrix to evaluatethe individual information(Ri)at each position in a sequence.For each evaluation, it also computesthe number of standard deviations away fromRsequence (Z score), and the one-tailed probability (p) of observing anormal splice site with that value of Ri.
Sequences with Ri valuesthat are either significantly greater or less thanRsequencehave low probabilities of belonging to the natural population of sites.A walker graphically shows the contributions of eachposition to a binding site.In the display(generated by Makewalker or Lister),favorable contacts between the spliceosome and a test sequenceare indicated by letters that extend upwards,whilepositions that are predicted to make unfavorable contactsare shown by inverted letters.Makewalker is interactive and shows one walker at a time, whileLister displaysmultiple walkers aligned with sequences and annotated by coding regions(e.g. Figs -).Selection of mutationsHuman splice site mutations were chosen from publishedreports for which corresponding genomic sequence data were available.Only a subset of reported mutations could beanalyzed, as sufficient intron sequences were often unavailable(&26 nucleotides for acceptor sites, &7 nucleotides for donor sites).To investigate the relationship between Ri value and splicesite use,studies that evaluated expression of the mutant mRNAwere selected whenever possible.A sequence interval (&100 nucleotides) surroundingthe splice junction was scanned to detectpotential cryptic splice sitesin the vicinity of the naturalsite.
Larger sequence windowswere used for cryptic sites known to occur further awayfrom the natural site (e.g. Table 2,#24).Two mutations could not be analyzed because there werediscrepancies at corresponding splice site sequences from different reports.A mutation in the IVS 10 acceptorof the hexosaminidase B genecould not be analyzed because the naturalacceptor site had negative information content in one of the sequences[,].A similar inconsistency was found in two different versionsof the IVS 5 acceptor sequence of the protein kinase C gene[,].Statistical analysesNatural and variant siteswith Ri& 0were compared withRsequence []by using the Z statistic and associatedprobability of observing a site with a particular Ri value[].Primary mutationsfor eitherdonor or acceptor siteswere analyzed by determiningthe average differences inRi values ()of natural versus mutantsequences.Significance was evaluated using a paired t-test.Mutations in which cryptic splicingwas either predicted or demonstrated experimentallywere excludedto avoid biasing estimation of,sincecryptic splicing can alter natural splice site usein the absence of a change inthe information content of that site.The observeddistributions of the locations of cryptic donor and acceptor siteswere compared witha model that assumes that these sites are equally likelyto occur upstream or downstream of the natural site.Significance was evaluated with thebinomial distribution.Relationship of information content to splice site useDifferent mutation reports measured splice site use directly by eithercDNA sequencing,reverse transcription-PCR,primer-extension, S1 nuclease analyses,or allele-specific hybridization.Direct comparisons of natural and mutant splicing patterns werenot always available.In some instances, the effect of the mutationwas measured indirectly using eitherNorthern hybridization(Table 1, #46, 47, 49; Table 3, #4),antigen immunoprecipation or protein levels(Table 1, #18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, 49;Table 2, #40, 41, 42, 43;Table 3, #2, 3, 4) ormeasurements of enzymatic activity(Table 1, #18, 19, 20,21, 23, 24, 25, 26, 27, 28, 29, 30, 34, 35;Table 2, #40, 41, 42, 43 ; Table 3, #2, 3).Functional analyses of splicingwere not reported for mutations#31, 32, 54 and 55 in Table 1,#14, 15, 23,34-38, and44, 45, 46 in Table 2,and#1, 7 and 8 (the natural site at 2621) in Table 3.RESULTSSeveral categories of mutations were distinguished byindividual information analysis. A total of 111 nucleotidesubstitutions were evaluated.Fifty-seven mutations were nucleotidesubstitutions that solely altered use of the natural splice site and did notcreate cryptic splice sites (designated as primary splice site mutations,Table 1).Activated cryptic splice sites were predicted for 46different mutations, 33 of which were corroborated experimentally(Table 2).Eight nucleotide substitutions were predicted not to alter splicing(Table 3).I. Primary mutations in splice junction recognition sequencesDifferences in information content of natural and mutantsplice sites.Many of the primary splice junction mutants that showed complete exonskipping(residual splicing: -)had Ri values bits(Table 1, #2, 3, 11, 12, 15, 16, 17, 19, 35).However, there are primary mutant donor andacceptor sites that were not usedthat have mostly small positive Ri values(Table 1, #4, 5, 14, 20, 21, 24, 36, 38, 40, 43, 47).This suggests thatrecognition of splice donor and acceptor sitesrequires more than zero bits.Mutations that reduce or completely abolish splicinghave significantly lower Ri values than the correspondingnatural sites.The average difference in Ri between primary mutant and naturaldonor sites isbits (n = 45)and for acceptor sites it isbits (n = 12),and these differences are significant(p&0.0001 for bothvalues).Ri values ofprimary acceptor mutations range froma minimum of -2.90 bitstoa maximum of 11.75 bits,whereas donor mutations have a lower range from-14.25to6.87bits.We considered the possibility that the strength of a natural splice site,i.e. Ri value,might be related to its susceptibility to mutationalinactivation.
15 of 24 (62%) natural sites in Table 1with Ri values& Rsequencewere inactivated by mutationor had mutant Ri values ,compared to 22 of 29 (76%) natural sites with Ri values& Rsequence.Inactivation of splicing is primarily determined by thespecific nucleotide substitutions that occur at those sites,however weak natural splice sitesmay be more susceptible than strong sites tosuccumb tomutations that abolish splicing.Amount of information required for splicing.The minimumquantity of information required for splicing, Ri,min, was definedby comparing the Ri values ofinactivating to leaky primary mutations(cryptic splicingmutations were excluded because activation of cryptic sites may affectnatural site use).Ri,minis bounded by themaximum information content of a non-functional siteand the minimum quantityof information required to produce normal transcripts.The following minimally functional sites had small positive Ri values:A mutation at the exon5 donor site (bits)in the HEXA gene results in a low level(3%) of normal mRNA (Table 1, #41).Similarly, a mutation at the exon 4 acceptor site(bits)in the APOE gene results in 5% ofnormal splicing(Table 2, #2)anda mutation at the IVS 14 donor site (bits)in COL1A1 decreases(by 50-60%)but does not abolish normal splicing(Table 1, #9).Furthermore, a mutant 2.4 bit acceptor site in the IDS gene(Table 2, #30) is associated with a moderately abnormal phenotype (the otherallele is null), consistent with production of somenormal mRNA.Finally,a mutation at the IVS 6 acceptor in COL1A2 reduces the Ri value ofthe splice site from 5.4 to 2.4 bits and results in a mild form ofEhlers-Danlos (type VII) syndrome due to 50% exon skipping (Table 1, #13;Fig. ).Splicing at this site is completely impairedin vitro at 39andrestored at 30.The temperature sensitivity of this mutation indicates that this 2.4 bitsequence is weakly bound by the spliceosome.By contrast, mutationsat the exon 1 donor splice sitein the CAT gene
(Table 1, #4;bits),in IVS 33 of COL1A2 (Table 1, #14;bits)completely abolish mRNA splicing.
The Ri value of this COL1A2 mutationis inconsistent with the result found for mutation #13, since the mutationwith lower information content would be expected to be inactive.This difference maynot be significant depending on the (unknown) precision of theRiw(b,l)matrix, however it seems more likely thatresidual splicing at the mutated site in mutation#14 may not have been detected.Residualsplicing was observed at several mutant splice sites with Ri valuesgreater than 2.4 bitsand less than 3.2 bits (Table 1, # 9, 41 and 52).These splice junction mutations define a range of valuesfor Ri,min of either donor or acceptor sites.Although the confidence interval around Ri,minis unknown, donor and acceptor splice sites with Ri&2.4 bits arerarely found in aset of random sequences with human dinucleotide composition (p=0.008).To simplify comparisons between Ri,minand other Ri values,we usebits.Leaky splicingTo determine whether the information present in a mutant sitewas related to splice siteuse, the Ri values of mutated splice sites thatinactivated splicing were compared withRi values of leaky splice sites.
Completely inactivatedsites generally hadRi values less than Ri,min (e.g. Table 1, #46), whereasmutations with Ri values greater thanRi,min reduced but generally did not abolish splicing.
For example,a GC point mutation in the exon 2 donor site of the LFA1 gene(Table 1, #44) decreased Ri from
8.6 to 4.2 bits and thismutation is leaky, i.e. 3% of the normal spliced productis detected from this allele[].Likewise, apatient with mild cholesterol storage disease
was homozygous for a donor sitemutation in the LIPA gene(Table 1, #45; Fig. ).Mutations #1, 6, 7, 9, 10, 13, 18, 22, 26, 34, 41, 44, 45, 50, 52, and 56(Table 1) and#2, 3, 4, 7, 9, 16, 21,23, 27, 28, 30, 32 and 41 (Table 2),which have Ri values,are leaky at the respective natural splice sites.The average decrease in Ri values is smaller forprimary mutations thatresult in reduced levels ofnormally spliced mRNA;isbits for donor sites (n = 12; versus -7.67 for alldonor sites)andfor acceptor sites (n = 4; versus -5.97 for allacceptor sites).When cryptic splice site mutations thatresult in residual splicing at the natural siteare considered in addition,the change is negligible:bits (n = 14) fordonor sites andbits (n = 15) for acceptor sites.Quantitative relationshipThe quantitative relationship between splice site useand information content is illustrated by thepolymorphic alleles in IVS 8 of the CFTR gene(Table 1, #6; Fig. ).The frequency of exon 9 skipping is inverselyrelated to the length of the polypyrimidine tract ofthe upstream acceptor site[,,].This is not surprising sincethe length of a homopolymericpolypyrimidine tracthas also been related to splice site strength [].The 4.1 bit difference betweenthe Ri values of the shortest and longest alleles accounts for thelower amount of spliced mRNA from theshorter allele and is probably related to the phenotypeof congenital bilateralabsence of the vas deferens in male homozygotes.A 4.1 bit reduction in informationwould correspond toat least a 17 fold()decrease in splicing,assuming minimal conversion ofinformation to energy dissipated[,].This corresponds closely tothe relative amounts of mRNA produced by theshortest (5T) andlongest (9T)alleles [].Only two exceptional mutations were found in which,althoughthese siteswere reportedly not used (Table 1, #5 [11.6 bits], #43 [5.7 bits]).The minimum predicted decreasesof3 and 11 fold, respectively,in binding affinitywould not be expected to completely abolish splicing at these sites.Reduced amounts of splicing canoccur at mutant splice sites withRi& Ri,min, although a modest decrease in Ri at a splice site canapparently sometimes inactivate splicing.II. Detection of cryptic splice sitesCategories of cryptic splice sitesRi analysis detected secondary crypticsplice sites that areactivated by mutation in or adjacent to the natural primary splicesite.This indicates that the Ri valuesof activated cryptic sites may be determinedwith an information model derived from natural splice sites[].Table 2 shows 33 experimentally-identified cryptic sites confirmed byinformation analysis of the respective genomic sequences (section A),and 13 mutations that were predicted by Ri analysis to exhibit cryptic splicing(section B).For example, a mutation at position 35066 of the adenosine deaminasegene(Table 2, #1)does not alter the Ri value of the natural splice site(at 35099),but creates a secondary cryptic site of similar strength at position 35067.There were 7 additional mutations in which a new cryptic site waseither created orpredicted without altering the Ri value of natural splice site(Table 2, #12, 14, 15, 26, 31, 40, 43).Activation of cryptic sites can also prevent splicing at natural sites bypromoting exon skipping (e.g. in 79% of transcripts resulting from amutation in the iduronate-2- Table 2, #26;[]).Exon skipping mutations occurred predominantly atdonor splice sites (7 of 8) and in each instance,a cryptic site was created upstreamwhose Ri value exceeded or was similar to that of the natural site.Several types of cryptic splicing mutations were distinguished:1.The most common category(n = 17)showeda concerted increase in information at the cryptic site(bits)accompanied by a reduction in the Ri value at thenatural site(bits).All of these were acceptor sites(Table 2, #7, 23, 25,27, 29, 30, 32, 34, 36, 37, 38, 39, 41, 42, 44, 45, 46).Thedistance between these cryptic and natural splice sites is, on average,4.3nucleotides, which would be expected for a mutation thatsimultaneously alters theRi values of both sites.
Detection of cryptic sites that overlap thenatural site requires sequence analysis of the mRNA, sincechanges in the size and sequence of the processed transcript are subtle.Use of these cryptic sites would either alter the reading frame orinsert or delete one or more codons(e.g. Fig. ).2.Novel cryptic sites were createdsimultaneously with eithermissense mutations(Table 2, #4, 12, 14, 15) orsilent coding substitutions (Table 2, #31).By creating a cryptic site,some of these coding sequence substitutions(Table 2, #35, 36, 37, 38, 40, 43)couldalsoinactivate the natural splice junctionor causeframe shiftinginstead ofexon skipping.Cryptic sites that generate mRNAs with in-frameinsertions or deletionscan also be recognized by Ri analysis[].3.Mutations that decreased the Ri value of the natural siteresulted in the use of pre-existing cryptic sites with Ri values in thenormal range(Table 2, #2, 3, 9, 10, 11, 13, 17, 18, 19, 20, 21, 22, 24, 28, 33).Some residualsplicing may occur at a mutated natural sitewhen the sequence change producesmutantand cryptic sites with similarRi values (e.g. Table 2, #7).Natural and cryptic sites competewith each other[,]when the natural siteexhibits either a moderate or no reduction in Ri.Susceptibility to activationOf 31 experimentally-verified cryptic splicing mutations(Table 2A, excluding #5 and 6),there are 19 splice siteswhose Ri values exceeded the cryptic siteprior to its activation(bits).For the remaining 12 mutations(10 of which involve the same site in HBB),the inactive cryptic sites exceedthe natural site by only anofbits.Furthermore,the differences in Rivalues between natural and cryptic sites prior to mutationalactivation are muchsmaller for donor sites(,n = 17 for donors vs.,n = 15 for acceptors).Likewise,cryptic donors were activated by an increase ofbits (n=5),whereascryptic acceptor sites were activated bybits (n=10).From these observations it would appear thatdonor sites may be more susceptible tothe effects of neighboring cryptic sites.Distance effectsCryptic sites activated by a mutation that weakensthe natural site must residewithin a few hundred nucleotides of the natural splice site, sincethe novel exon is restricted in length[,].For example, a strong cryptic acceptor in intron 2 of the-hemoglobin gene isactivated by mutations at the exon 3 acceptor 271 nucleotides downstream(Table 2, #24).Mutation at a natural site can, however, activate sites that arefurther away when a cryptic exon is created.For example,mutation at the exon 3 acceptor of the CFTR gene activatesa cryptic, non-coding exonin intron 3 (2,354 nucleotides downstream of exon 3 and 19,329nucleotides upstream of exon 4;Table 2, #3).ExceptionsAlthough pre-existing or novel cryptic sites with Ri valuesless than that of the strongest local splice site were usuallynot recognized, there were exceptions.Infrequently, a weaker cryptic site can interfere with a naturalsite, even when the natural site is strengthened by the mutation(e.g. Table 2, #16).For example,activated cryptic sites with Ri values lower than those of thenatural splice site after mutationmay sometimes be used(Table 2, #1, 3, 4, 6, 9, 16, 23, 32).In at least one instance(Table 2, #1),a cryptic acceptor site upstream of the natural site ispredominantly useddespite the fact that both siteshave similar Rivalues, which suggests that the cryptic site is recognized first.Conversely, the Ri value of the exon 1 donorin the -globin geneis less than that of an upstream cryptic site(Table 2, #12-15, 17-22),however this cryptic site is not activated unlessit is strengthenedorthe donor is weakened.These exceptions suggest thatbesides directcompetition between the cryptic andnatural splice sites,other factors can influence splice site selection.Another class of exceptional splice sites werethose that generated alternatively processed transcripts.Active ``cryptic'' sites that resided in introns of the CSPB gene hadRi values in the normal range(Table 2, #5, 6)[,].They may represent alternative splice sitesregulated by other sequence elementsthat can be present in the adjacent exons[,,,,]or polypyrimidine tracts[,].III.
Non-deleterious splice site substitutionsNucleotide substitutions that do not significantly alter the Ri value of anatural site are expected to produce functional rather than mutant sites[].Giventhat such substitutions are not likely to be deleterious, they may bepolymorphic in the germline, as has been shown for a sequence change in anhMSH2 splice acceptor site [].We identified other nucleotide substitutions that did notsignificantly alter the Ri value(Table 3):1.Reported mRNA analyses of substitutions #4 and 5 did not reveal splicingdefects that altered either the size, structure or quantity of thesetranscripts although these changes had been suggested to affect splicing[,,,,].2.A CG substitution 12 nucleotides upstream of theIVS 2 acceptor of the CYP21 gene(Table 3, #6)decreases in Ri value byonly 1.56
bits and mRNA of normal size and quantity was present[].Asymptomatic individualswith this sequence havebeen reported [,,]and a comparableresults from abenign CA polymorphismat the same position (Table 3, #5).3.An AG substitution at the exon 7 donor site of the OTC gene wassuggested to cause exon skipping,however Northern analysis did notshow either the size or quantity of mRNA to differ from controls and thechange in Ri was negligible (Table 3, #4).Since OTC protein was not detected,this patient may harbor amutation elsewhere.Splicing patterns for several nucleotide substitutions#1, 2, 3, and 7 (Table 3)were not reported, however, based on information analysis,these changes would not be predicted to alter mRNAsplicing.The substitutionseither maintain orincrease the information content of the natural splice site. The Ri valuesof the proposed cryptic sites for substitutions #1, 2, and 8were either negativeor unchanged, suggesting that they are not activated by thesesubstitutions.
A proposed cryptic site in exon 3 of the p53 gene(substitution #7)is significantly weaker than the natural acceptor site(by 6.14 bits)and has an Ri value only slightly larger than Ri,min.It would seem unlikely that this cryptic site is preferentially used.DISCUSSIONThe number of bits in a splice siteis related to the amount of splicing at that site.Previously, we demonstrated that apolymorphic splice junction variantcaused little change ininformation[].The present study extends this finding andshows that mutant splice sitesoften contain significantly less information than their corresponding naturalsites.Further,cryptic splice sites are activated by increases in informationor by decreases at the natural splice site, andthe information atactivated cryptic sitesis often comparable to or exceeds the natural site.Predicting the effects of mutations A required step of information analysis isto compute the total information over all positions in a site.This value must then becompared with that of other sitesprior to concluding that a substitution thatchanges a positive to a negative weighting is deleterious(compare Tables 1 and 2 to Table 3).Functional splice sites can havenucleotides with negative weightings(e.g. Fig. , position 63)that are offset by strong contributionsat other positions(e.g. Fig. , position 64),as we have shown for other binding sites(Figure 2 in Schneider.walker, Hengen.fisinfo).Statistical analyses of the distributions of pointmutations in splice sitesare useful[]but can sometimes obscure these compensating effects.Within a binding site,the context of a mutationcan be as important as the mutation itself.The difference between the observed value of Ri,min (bits) and itsexpected value (zero bits) may have a biological basis.However,this difference could also be explained by errorsin the database used to create thesplice weight matrices [],statistical limitations of the data and matrices,motifs that are different from themajority of sites [],orintrinsic limits to the precision of splice site recognition[].Although the standard deviation ofRsequence can be determined [],the confidence intervals on individual Rivalues are unknown.These intervals are expected to be largerat thelowerandupperbounds of the Ri distribution,where fewer functional splicesites are observed.The existence of a natural site withRi& Ri,min(2.2 Table 2, #26)and an exon-skipping mutation withRi& Ri,min(3.2 Table 1, #14)suggests thatRi,min is not known precisely.The error(|Ri- Ri,min|)may beas little as 0.2 bits(Ri= 2.2 Table 2, #26),but it might beas much as2.4 bits(Ri= 0 Schneider.Ri).Susceptibility to mutation Donor sites may be moresusceptible to inactivation than acceptor sites.The Ri values of mutant donor sites aremore likely than mutant acceptors to beless than Ri,min.Natural donors possess less informationthan acceptors[]and the average decrease in information due to mutation at donor sitesexceeds the reduction in Ri rmation is also less densely distributed across acceptor splice sites(0.3 bits per nucleotide) than in donor sites (0.8 bits per nucleotide),so changes at acceptors often have a smaller effect on Ri.Significantly more primary mutations in donor sites (n=45) thanacceptor sites (n=12) werefound, as has been noted [,].Cryptic splicing The Ri values of most novel cryptic donor sitesexceeded or were similar to those of the corresponding natural sites. Althoughsimilar results were also inferred from Shapiro-Senapathy consensus values[], information analysis detects fewer incorrectcryptic splice sites [],more accurately discriminates true sites fromnon-sites, and visually depicts both changes (Fig. ).An exon is initially defined by recognizing the acceptor[].Crypticacceptorsites occur eitherupstream (n = 9)ordownstream (n = 7)of the natural site(p = 0.4),suggesting that they are not located by scanning[].The exon definition model predicts thatthe spliceosomethen scansdownstream until a strong donor site is located[,],soa novel cryptic donor site createddownstream of an intactnatural site should not be recognized unless thenatural site is mutated.In all cases,a decrease in the information content of the natural donor siteactivated pre-existing cryptic sites downstream(Table 2A).Furthermore, cryptic donor sites were activated morefrequently upstreamof the natural site(15 of 20; p=0.02).The idea that the splicing machineryselects for the strongest local acceptor splice siteand scans for donorsis supported by Ri analysis.Nucleotide substitutions within 17 natural acceptor siteshave been shown to create or strengthen adjacent cryptic sitesthat are thereby activated(see results: II. Detection of cryptic splice sites).Only acceptors were found,perhaps because the variablepolypyrimidine tract potentiates spliceosome recognition at many positions,whereas donor sites have high information densityand a non-repeating sequence pattern[].For this reason,weaker cryptic sites are often found near natural acceptor sites(e.g Fig. ).Mutations involving the natural acceptorsometimes strengthen and activate these cryptic sites.The resulting aberrant exons may in some cases have beenmisidentified as natural splice products(e.g. Table 2B),since their length and sequencewould differ by only a few nucleotides from the normal mRNA.ConclusionWe have shown that individual information theory can be usedto rank normal and mutant splice junctions.As a consequence, silent polymorphismscan be distinguished from true mutations,changes in individual information are related to splice site use,and activated cryptic splice sites can be detected.These distinctions are possiblebecause the information measure is related to the thermodynamic entropy, andtherefore can be connected to the binding energy[,,,]. Theinformation in the splice site should be related to the specific bindinginteraction between the spliceosome and the site[,,,].However, the relationship is aninequality--the second law of thermodynamics[,]--andcan only be explored empirically at this stage.The correlation between informationmeasures andmeasured thermodynamic parameters is expected to more preciselyrelate genotypes to phenotypes in genetic disorders.ACKNOWLEDGEMENTSWe thankGreg Alvordfor statistical consulting,andKenn Kraemer andHoward Youngfor reading the manuscript.Grant support is acknowledged from the Public Health Service (CA74683)and the American Cancer Society (DHP-132) to P.K.R.We thankthe Frederick Biomedical SupercomputingCenter for access to computer resources and support services.21
Figure 1:A primary splice junction mutationrepresented by sequence walkers.A GA mutation 1 nucleotide upstream of the exon 6 donorof the COL1A2[GenBank accession number M35391]gene results in 50% exon skipping and Ehlers-Danlos syndrome,TypeVII(Table 1, #13).This substitution, which significantly reduced the Ri value, definesthe lower threshold of information required for splice site recognitionsince it is temperature sensitive,being non-functional at 39but functional at 30.The splice sites are shown by walkers[]in which the height of a letter is the contributionof that base to the total conservation of the site.The upper bound of the vertical rectangles is at +2 bits,and their lower bound
is at -3 bits.Letters that are upside down and point downwardsrepresent negative contributions.The upper walker sthe lower one displays the mutant sequence.The black arrow shows the position of the mutation (boxed).The dashed arrow represents the coding region. Figure 2:A leaky splice junction mutation.A GA mutation 1 nucleotide upstream of the exon 8 donorsite of the lysosomal lipase gene [LIPA; U04292] results in mild cholesterolester storage disease with 4-9% enzymatic activity(Table 1, #45).The reduction ininformation content is significanteven though the Ri value is still muchgreater than Ri,min. Figure 3:Polymorphic variation that affects splicing.Splicing varies among3 common alleles that differ inlength in the polymorphic polythymidine tract of the IVS 8 acceptor of thegene encoding the cystic fibrosis transmembrane regulator [CFTR; M55114](Table 1, #6).The shortest allele (bottom walker) shows 90% outsplicing of exon 9and is associated with congenitalabsence of the vas deferens.Individuals with the two longer alleles havea normal phenotype, although the 7T allele producesless mRNA than the 9T allele.Exon 9 begins at the base indicated by the left bracketand dashes. Figure 4:Cryptic site creation concurrent withmutation of the natural site.An AG mutation in intron 3 of the iduronidasesynthetase gene [IDS; L35485] significantly decreases the information contentof the IVS 3 acceptor whilesimultaneously creating a strong cryptic site at theposition of the mutation, 1 nucleotide upstream from the natural splicejunction(Table 2, #27).The upper two walkers show a pre-existing crypticsite at position 5153 anda natural site at 5154.The lower two walkers show the activatedcryptic site at 5153 andthe mutant site at 5154.For simplicity,only sites with greater than 4.3 bits are shown.In addition, a 4.2bit sitethat is not usedat position 5155,is reduced to 2.5 bits as aconsequence of the mutation.The lower bound of the vertical rectangles is at -7 bits.
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