23 ~- -

Cytogenetics and Genome Analysis Wirh thegenomes ofh&her oyanisms being organized into structural af:d finctionnl erztities - chromosomes - it is essential that DNA sequerxe be related to chromosomal organizatiorr. The reverse side of this coin is (hat a good tmderstattdirrg of chromosome slructure and

jrmnction provides tooisforgenome mapping and analysis. This section reviews two areas ~Jcytogenefics relevant to genome analpis which have seen major advances recently: FISH (‘~orescence in-situ hybridization); and the use ~fjirnctional genetic elements for the isolation and~irnctional studies ojgenes, including the construction of artificial chromosomes - YACs and MACs.

Using chromosome features in genome mapping

--_-____Howard Cooke

Studies c? the structure and function of chromosomal features and sequence el-

ements have much to contribute to and gain from genwne mapping. AbnormaRies

in chromoscme banding cissociated with specific diseases have pinpointed regions

of the ~~KNW for intensive analysis. An improved understanding of chromosome

organization can facilitate gene identification and isolation, as well as enabling

sophisticated genetic and vector systems for genome studies to be devised.

Genomc mapping is a process that places points (gcn- eric marken or DNA sequences) in a line which. in both genetic and physical tcrn~s, rcprescnts the chro”- nxxon~ In higher eukri~otcs the map is a straight line, reflecting the fact chat the DNA n~alccules and the chronlosonxs arc alqo linear.

Funclional chromosomal &ment~ In addition to the requiremc.nr for histonc and non-

histonc proteins, ;wd RNA. nxxintcnancc drhe DNA ax a stable, linear rcplicatin, ir chromozon~ in higher cukayores requires the prescncc of three DNA sequclxe clcmcnts: a re$ication ongn. a c~ntromerc’ and telomeres.

The need for telomcrcs {ix. 5iIllpk rcpcatcd

sequences at each end ofthc chronlos~t~~lc~j iz imposed by- chromosome lineatiy: they ensure complete rcpli- cation of the chromosomes, and protectinn i?onr fusion at the chromosome ends. Trlomcws ah i:ro- vidc absolute r&rcnce points &r gcnctic lnd ph?isal ~ntlp~: ~IIIICSS thr ntap itlcl&i both teiomcrcs of a chronwSomc thcrc is no guarantee char it is cony~tt~rc.

Box 1. Chromosome-banding techniques

Staining chromosome: rev& banding patterns that are related to the structure and composition of the chromosome (Fig. la). The unique banding pattern of individual chro- mc;;cimes permits ihem to be distinguished Born other chromosomes of similar size.

Giemsa and orcein dyes produce uniformly stained chromosomes. However, partial

a

digestion of chromosomally associated proteins with trypsin followed by staining with Giemsa gives rise to dark bands I’G’ bands). These bands are almost identical to the pat- tern obtained @’ bands) on staining with quinacrine dye which binds preferentially to AT- rich regions (as does the dye Hoechst 33258) -under UV, these regions then fiuoresce. Reverse banding (‘R’ banding) involves chemical pretreatment of ihe chromosomes foF iowed by Giemsa staining-this results in preferential binding of the dye to GC-ricjl DNA, producing a banding pattern opposite to G banding. An alternati’:e pretreatment w&h acid and alkali, to remove -60% of DP’I from the chromosome, followed by Giemsa stain- ing, produces ‘C’ bands (constituiive heterochromatin) which correspond to the ten tromeres (Fig. 1 b).

7 Telomere region

An adaptation of staining techniques involves the incorporation of bromodeoxyuridine (ErdU) in place of thymidine during replication: BrdU-containing DNA stains differently. BrdU substitution followed by Giemra or Hoechst staining can be used to investigate which regions of chrumosomes are replicating actively, since not all regions replicate simultaneously.

Centromeie region

3 Telomere region

Figure 1 (a) Basic chromosome structure. (b) Human chromosome 3 banding by different staining methods: (i) G banding; (ii) Q banding; (iii) 4 banding; and (iv) C banding (see BCJX text). Note that (il and (ii) have essentially the same pattern, but reversed. The heterochromatic region, sttongly stained in (iv). stains only weakly by the other procedures.

ii) (ii) (iii) 0”)

hman chro~noso~~~cs. These fcaturcs, as well as hav- ing structural significmce, have provided useful land- marks for mapping pur~osss, III conjunction with irr sillr hybridization, the banding pattern cm locate a DNA sequcncr to a particular region ofn cl~ro~noson~c

with a resolution of about 5-10 Mb. Altcrcd banding pattc‘rns for a particular chromosome allow breakpoints and dclctions in chromosomes to be ass&cd to par- ticular regions of the genon”. In favourablc cases this can be used to done genes associated with these brcak- points.

Cytogenic bands retlcct a levc! of scqucnce organiz- ation which is beginning to enlcrgc front long-range mapping and an increased knowledge of ~CIIOIIIC organization. Such organizational features nlcludc repeat scqucnccs, bo;.h dispersed and tanden], in ad- dition to features such a5 the ccl:troinerc and tcioimcrc.

Repeat sequences AIIKMI~ the mnny repeat ~qucnccs now recognized

thcrc arc two connmon intcrspcrscd rcpeatcd LJNA scqucnccs (IRS) of ~~nlmow~~ functions in the human gcnornc’; the Alu t%mi!y (about IO” copies present) end the Kpn bnlily (about IO” copies ofthe niost abundant, 3’, end), nnmcd after the restriction cnzyms sites rmost characteristic of thetm. The availability of spccics- specific PC11 ~polyiiicrare chain reaction) priniers for these repeats has allowed probes to bc generated3 from, for instance, regions of bu~mn chro?mosormes flanked by Alu repeats contained in interspecific cell hybrids

(e.g. II-~~~~~-~LIIII~II hybrids frorm which most of the human chromosomes arc lost). hr sitar hybridization with an Alu probe gives rise to a banding partcrn, cssen- tially the reverse of that given by the Kpn &lily which produces a signal corresponding to the dark ‘G’ lxds'. This non-randcnn sequence distribution is also ref- lcctcd in another sequcncc feature which has known biological irxportancc in vertebrate genon~ - CpG islands.

Genes, island’s and restriction maps

In vertcbratc DNAs, the frcqucncy of the di- nucleotidc CpG is Iowcr than would bc cxpcctcd on the basis of base conlposition. However. some regions of nlanlnla;ian and other gcnomcs arc not reduced in CpG contentS; thcsc regions are short (-1 kb) and associated with the 5’ ends of many gxcs. Thcsc ‘CpG islands’ are usual!y uimmethylated. whereas CpG d~nuclcotidcs occurring oursidc the islands are very ficqucntly mcthylatcd and associated Witli specific proteins. This, and the absence of island-associated nucleoson~es ard histonc El, suggests the islands rmay rcprcscnt 'open regions ofthe gcnomc CO which other protcii:s, such as transcription r’actors, nray have rcla- lively easy ;ICCCSS”. They also facilitate the generation of large-scale restriction nlaps of the gcnonlc: many restriction cnzy~ncs with C&G-rich recognition scqucnccs &xc mamniahan gcnomes at the CpG iciands, thus enabling the rapid idcntificaticn of puta- tive genes. Restrictiorl-ellz)mIc rnapping of islalida indicates their distribution is non-randon), with abrupt transitions between island- (and putatively gene- ) rich

TIET=SHJAN/FEB 1992(VOLlO)

35

cytogenetics

a Ml

t

Ml Ml

Cen t

I

Cen Cen Recombination Cell division

I

I t M3

M2 I s M3 M2

Figure 2 la) A generalized scheme for chromosome breakage and mapping with telomeres. Recombination behveen a telomere vector and a chr@ mosome followed by selection for the teiomere vector marker M3 can result in formation of a new telomere. If other markers are available (Ml and M2) then one can be used to retain the chromosome (Ml) whilst the uther (M2) can be used to detect or select against cells which, after cell division, retain ihe part of the chrrjmosome which should have been broken to give a fragment without a centromere. (b) Chinese hamsterchromasomes (il before and (ii) after creation of a new Clomere using a telomere/selectable marker consti-act. Chinese hamster DNA from the integration site of the vector was hybridized in situ to the parent ccl! line N and to the transformed cell iiil. The chromosome broken and the products of breakage are arrowed.

and -poor areas of the gwo1~~4. Evidence that the fragiic site (a rcgijon at which the chromosome appcxs cstcLJded or broken wiiis1J is associated Lvith the ColJllllOticSt X-ii&cd mcntai rctarclacion Syn- drouxe) on :he hurua~J X chronJoson,c is anociatcd with ablloruiai metiJyintion of a CpG islarld suggese that islands and nxthylation cau affect c:JronJoso:nc structure7. Whcthcr transitions bctwccn light and dark bands, or Aldrich and Kpn-ric5 Lands, correspond tc island (gcnc) rich and poor regions is unknown, but may hcip ciucidatc physical anA gmetic orgallization. Large-scaic ~,~~OJJJC mapping should provide the answer.

Using functional sequences for mapping FurJctioxJai clcments can be applic~i in several ways

to f3cilitate gene-isoiatiou and chromosomr-ti.l:lctioJJ studim. Wowwcr, although dcailcd I;nowlcdgc at the sequence Icvri of the functional ciculrnts ofciiromo-

iWamma/iun centromrres The characteristics of rnammalinn ccntromcre. arc

poorly utidc~srood; inlinunocytochcmii:nI cvidcncc indicates that they have many protein components”. Cytological studies show that tnndcm arrays of repcat DNA occur at thr ctx!tronIcrc, in blocks ranging from a few hundred kb to scvcral Mb. O~!L’ hunxm re- peat scqucncc (tbc alphoid :equcnce) ami its n~ousc counterpart both contain a 17 bp motif recognized i11 vitm by a ccntromcric protcirt. CENI’U”‘. Anti- bodies to CENPB inhibit chrcmmsomc‘ movcmcnt irr 1&011. Whcthcr or not thcsc scqu~~cs constitute actunl ccntromcrcs, they hnvc been ~4 for physical and grnrtic iiiapp& As with most rcpcnt scqucncos, thcrc is sutficictit copy-to-copy varintion that chro- mosonx-specific ccntromcric marker probes GUI bc

grncratcd from them.

Tclorncrcs of cukaryotcs xc rcl;ttivcIy well tmdcr- stood’?, largely because in all these org:misnrs (with a few cxccptions, notably Dn~q~lrilil) they consist of short, tx~dcmly repcatcd scqucnces with a G/C strand bias. Tclon~rcs from nl:ul~ JitTcrent organisms cnn function in yeast, and lmnan tclomcrcs have been cloned in this way. The adjacent scy~~e~xxs provide useful markcn for the ends ofchromosomcs wbicb can be used to gcncntc long-range restriction maps by par- tial dig&n and end Inb+rilirrg The tclomcric regions ofmany guwn~cs arc polymorphic, probably as 3 result of csch:mgc bstwcen chromosomes. The terminal rrpcats Arc’ subject to variation in number bctwcctl species a11d tissues a1111 arc shortuncd with age in an individual tissue, arid also in sotnc tunloi~rs’3~ 15.

When rc-introduced, cloned hutnm tclomcrcs em fUnction in nlnninialian cells 1’). Although the ~uxhan-

iwl is iuiJcar, the resulting chroniosoinc \vith a new tclomcrc is dclctcd for l)NA srq~~wccs lxtwecn this new telomcrc and the original tclomcrc. This suggests that they can bc used a~ chroiiiosotnc-brcnk;lgc tools for Illappiilg as has bucn discussed abovc for yeast

telomcrcs. Sets of ctxomosonics producd in this way

arc particuldy suitnblc for long-rang physical niap- ping bcca~~se end-lrbcllcd, *lx-tial-dig&on products GUI bc applied using the same probes for all chromo- SOtnCS.

Dcvclopmcnt ofa nxniimalian artifici:il chrolllo~omf (MAC) system (I\. Anand 1~ ii/., op. cit.) will bc cssc’n- tial f6r &sting the role of di&rcnt I)NA scqucnccs as rcplirntion origills, ccntronicrcs and pcrh:ips scatliold :lttachmUnt regions. MACs may also find a LISL' in gcnc therapy ifthcy Linction as non-integrating, copy-nuni- bcr-controllcd vectors. Obtaining tclomerc function frotn cloud DNA scqul:nccs may provide a step towards a MAC. If DNA can bc maintain,xl as lin<ar tnolcculcs tbcn, in principle, jicnctic systems can bc dcviscd to sclcct for CL’iltroinCrc and rcplicati:x Cunc-

tions, and deletion assays used to fttrthcr dchc the inprtant DNA SC~LIWCC~.

TIETECH JAN/FEE :992 (VOL 10)

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