Molecular Evolution: Producing the Biochemical Data

720 Pages·1993·13.234 MB·English

by  Abelson J.N., Simon M.I., Zimmer E.A.

Contributors to Volume 224
elcitrA numbers are in parentheses following the names of conUibutors.
moifailfffA ~ era .tnenuc
NYLIRRAD G. THGIRBLA (21), Laboratory of LEAHCIM J. BRAUN (1, 21), Laboratory of
Molecular Systematics, National Museum Molecular Systematics, National Museum
of Natural History, Smithsonian Institu- of Natural History, Smithsonian Institu-
,noit ,notgnihsaW D.C. 20560 ,noit ,notgnihsaW D.C. 20560
MARC W. ALLARD (34), Human Genome ENAID M. EGDIRB (4), Department of -loiB
Center, University of Michigan, Ann ogy, Yale University, New Haven, Con-
,robrA Michigan 90184 tucitcen 11560
JEAN-PIERRE EIRELLEHCAB (25), Labora- RoY J. BRITTEN (17), Kerckhoff Mar-
toire de Biologie Mol~culaire ,etoyracuE ine Laboratory, California Institute of
Institut de Biologie Cellulaire du CNRS, ,ygolonhceT Corona del Mar, ainrofilaC
31062 ,esuoluoT France 52629
MARK A. BATZER (16), Human Genome JANICE BRITTON-DAvIDIAN (7), Labora-
,retneC Biology and Biotechnology Re- toire de Genetique et Environnement, In-
hcraes ,margorP Lawrence Livermore Na- stitut des Sciences de L'Evolution, -vinU
tional Laboratory, Livermore, ainrofilaC Misre Montpellier II, 34095 ,reilleptnoM
15549 ecnarF
YROGERG C. UEILUAEB (18), Department of LORAC J. BULT (6), Laboratory of raluceloM
,ygoloiB Western Washington ,ytisrevinU Systematics, National Museum of Natural
,mahgnilleB Washington 52289 History, Smithsonian Institution, Wash-
DLONRA J. HCIDNEB (18), Department of ,notgni D.C. 20560
Botany, University of ,notgnihsaW ,elttaeS ACCESER L. CAm~ (3), Department of -eG
notgnihsaW 59189 netics and Molecular Biology, University
MEREDITH LLEWKCALB (5), Department of of Hawaii at ,EGnaM Honolulu, Hawaii
,ynatoB Louisiana State ,ytisrevinU Baton 22869
Rouge, Louisiana 30807
Rose NNA OCILOTTAC (13), Department of
HTIDUJ A. EKALB (I), Laboratory of -celoM Botany, University of ,notgnihsaW ,elttaeS
ular Systematics, National Museum of ,notgnihsaW 59189
Natural History, Smithsonian Institution,
,notgnihsaW D.C. 20560 SHENG-YuNG CHANG (32), Roche Molecu-
lar Systems, Inc., Alameda, ainrofilaC
ALLENURB MARTIRE HCTIDWOB (21), Lab-
10549
oratory of Molecular Systematics, Na-
tional Museum of Natural History, Smith- LLESSUR L. CHAPMAN (5), Department of
sonian Institution, Washington, D.C. Botany, Louisiana State ,ytisrevinU Baton
06502 ,eguoR Louisiana 30807
YHTOMIT NEWOB (38), Department of -eG JOBY MARIE KCINSEHC (13), Department of
netics, University of Leicester, Leicester ,ygoloiB Lafayette ,egelloC Easton, Penn-
LE1 7RH, United Kingdom sylvania 24081
ARAaRAB H. NAMWOB (29), Roche -uceloM RoE ELLASED (4, 14), Department of -otnE
ral Systems, Inc., Alameda, California mology, American Museum of Natural
10549 History, New York, New York 42001
ix
X CONTRIBUTORS TO VOLUME 224
TTOCSERP L. REGNINIED (16), Department ical ,secneicS Australian National -revinU
of Biochemistry and Molecular ,ygoloiB sity, arrebnaC City, ACT ,1062 Australia
Louisiana State University Medical DRAHNREB AUER (44), BASF, Aktienge-
,retneC New Orleans, Louisiana ,21107 ,tfahcslles ZHB-Biotechnologie, D-6700
and Laboratory of Molecular ,sciteneG ,nefahsgiwduL Germany
Alton Ochsner Medical ,noitadnuoF New DIVAD M. HILLIS (34), Department of -looZ
,snaelrO Louisiana 12107 ogy, The University of Texas at Austin,
WEHTAM DICK (4), Department of ,ygoloiB Austin, Texas 21787
Yale ,ytisrevinU New Haven, tucitcennoC
KENT E. REGN1$LOH (33), Department of
11560
Ecology and Evolutionary Biology, Uni-
LEIRBAG A. REVOD (38), Department of -eG versity of ,tucitcennoC Storrs, tucitcennoC
netics, University of Leicester, Leicester 96260
LE1 7RIt, United modgniK
NHOJ A. HUNT (23), Department of sciteneG
LEINAD E. NEZIUHKYD (45), Department of and Molecular Biology, John. A. Burns
ygolocE and ,noitulovE State University of School of Medicine, University of Hawaii
New York at Stony Brook, Stony ,koorB at Manoa, ,ululonoH Hawaii 22869
New York 49711
DIVAD M. IRWIN (40), Department of -inilC
WERDNA D. NOTGNILLE (47), Department lac Biochemistry, and Banting and Best
of Chemistry, Indiana ,ytisrevinU Bloom- Diabetes Centre, University of ,otnoroT
,notgni Indiana 50474 ,otnoroT ,oiratnO Canada MSG IL5
TREBOR A. NAMDLEF (3), Department of TREBOR K. NESNAJ (33), Department of -oB
Genetics and Molecular Biology, -revinU ,:)nat The University of Texas at Austin,
sity of Hawaii at Manoa, Honolulu, Austin, Texas 21787
Hawaii 22869 EUS NOSTREBOR-SKNrJ (46), Department of
INGRID REGLEF (23), Papua New Guinea In- ,ygoloiB Emory ,ytisrevinU Atlanta, -roeG
stitute of Medical Research, Madang, gia 22303
Papua New aeniuG ELORAC NOSSIOJ (10), Laboratoire d'Im-
DRANOEL A. FREED (3), Department of Zo- munochimie, Institut de Biologie -uceloM
,ygolo University of Hawaii at Manoa, laire et Cellulaire, CNRS, 67084 Stras-
,ululonoH Hawaii 22869 ,gruob France
WEHTTAM GEORGE (14), Department of NODLE R. JUPE (39), Department of Mole-
,yrtsimehcoiB Howard ,ytisrevinU Wash- cular Genetics, University of Oncinnati
,notgni D.C. 95002 College of Medicine, Oncinnati, Ohio
SAMOHT J. HSINVIG (2), Department of -oB 76254
tany, University of Wisconsin, Madison, NERUAL N. W. NAGROM-MAK (36), Divi-
nisnocsiW 60735 sion of Biochemistry and Molecular -loiB
DIVAD NAMDLOG (8), Laboratory of -orueN ogy, University of ,ainrofilaC Berkeley,
,sciteneg National Institute no Alcohol ainrofilaC 02749
Abuse and ,msilohoclA National Institutes YuN-Tzu KJANO (6), Department of Plant
of Health, Bethesda, Maryland 29802 ,ygoloiB University of New Hampshire,
MAILLIW J. HAHN (2), Department of -oB Durham, New Hampshire 42830
tany, University of Wisconsin, Madison, JACK F. KIRSCH (36, 42), Division of Bio-
nisnocsiW 60735 chemistry and Molecular ,ygoloiB -revinU
YRRAB G. HALL (44), Department of -loiB sity of ,ainrofilaC Berkeley, ainrofilaC
ogy, University of Rochester, ,retsehcoR 02749
New York 72641 SAMOHT D. KOCHER (28), Department of
NHOJ M. KCOCNAH (38), Molecular -orueN ,ygolooZ University of New Hampshire,
ygoloib ,puorG Research School of -goloiB Durham, New Hampshire 42830
CONTRIBUTORS TO VOLUME 224 xi
SUTELC P. KURTZMAN (24), Microbial SAMOHT D. PETES (46), tnemtrapeD of -loiB
seitreporP ,hcraeseR National Center for ,ygo ytisrevinU of North ,aniloraC lepahC
larutlucirgA noitazilitU ,hcraeseR -lucirgA Hill, North aniloraC 27599
tural hcraeseR ,ecivreS United States -eD
ELLEN M. PRAGER (11), Division of Bio-
partment of ,erutlucirgA Peoria, Illinois yrtsimehc and raluceloM ,ygoloiB -revinU
40616 sity of ,ainrofilaC Berkeley, ainrofilaC
YELRIHS KWOK (32), Roche raluceloM Sys- 02749
tems, Inc., Alameda, ainrofilaC 10549 LIANO-Hu Qu (25), Biotechnology Re-
SAMOHT B, LAVOIE (36), sreyM-lotsirB hcraes Center, Zhongshan ,ytisrevinU
Squibb lacituecamrahP hcraeseR ,etutitsnI uohzgnauG 015 275, People's Republic of
,notecnirP New Jersey 44580 anihC
EUQIRNE P. LESSA (31), Laboratorio de A. LANE RAYBURN (15), Department of
,n6iculovE lnstituto de ,aigoloiB Montevi- ,ymonorgA ytisrevinU of ,sionillI ,anabrU
oed ,00211 yaugurU Illinois 10816
SORDNA RUm LINARES (38), tnemtrapeD of LORAC A. Rime (3), tnemtrapeD of sciteneG
,sciteneG Stanford University Medical and Molecular Biology, University of
,loohcS ,drofnatS ainrofilaC 50349 Hawaii at Manoa, Honolulu, Hawaii
J. KoJI MUL (3), tnemtrapeD of sciteneG and 22869
raluceloM Biology, University of Hawaii ARABRAB ReIm.tOLD-HuREK (35), -trapeD
at Manoa, ,ululonoH Hawaii 22869 ment of Biological ,secneicS Center for
BARBARA LUNDRIOAN (37), Museum of Zo- Molecular ,sciteneG State University of
ygolo and Department of ,ygoloiB -revinU New York at Albany, Albany, New York
sity of Michigan, Ann Arbor, Michigan 22221
90184 SCOTT O. ROGERS (18), Environmental
ECURB A. MALCOLM (42), Division of gurD ecneicS and ,yrtseroF Syracuse ,ytisrevinU
yrevocsiD and ,tnempoleveD Chiton -roC ,esucaryS New York 01231
,noitarop ,ellivyremE ainrofilaC 80649 NEVETS H. ROOSTAD (20), Department of
SANDRA L. MARTIN (22), Department of lacigoloiB ,secneicS ytisrevinU of -nicniC
ralulleC and larutcurtS ,ygoloiB ytisrevinU nati, ,itannicniC Ohio 12254
of odaroloC School of ,enicideM ,revneD NEVETS OREBNESOR (42), Division of Drug
odaroloC 26208 yrevocsiD and ,tnempoleveD Chiton -roC
RICHARD .B MEAOrlER (26), Department of ,noitarop ,ellivyremE ainrofilaC 80649
,sciteneG University of ,aigroeG Athens,
CARL W. DIMHCS (16), Departments of
aigroeG 20603
Chemistry and Genetics, University of
MICHAEL M. OTOMAYIVP~ (34), tnemtrapeD ,ainrofilaC ,sivaD ainrofilaC 61659
of ,ygolooZ ytisrevinU of ,adirolF -seniaG
JULIE F, FFOCENES (26), tnemtrapeD of -eG
,elliv Florida 11623
netics, University of ,aigroeG Athens,
NEHPETS J. O'BmEN (8), yrotarobaL of lariV aigroeG 20603
,sisenegonicraC National recnaC ,etutitsnI
National Institutes of Health, ,kcirederF ANDY SHIn (32), lnnovir ,seirotarobaL New
Maryland 10712 ,kroY New York 12001
ETNAVS PAABO (30), Department of Zool- PHOneS SHin (42), Division of yrtsimehcoiB
ogy, University of Munich, D-8000 Mu- and raluceloM ,ygoloiB ytisrevinU of -lraC
hcin ,2 ynamreG fornia, ,yelekreB ainrofilaC 02749
NEHPETS R. PALUMBI (29), Department of DAVID A. BUHS (35), tnemtrapeD of -igoloiB
,ygolooZ and Kewalo Marine ,yrotarobaL lac ,secneicS Center for raluceloM -teneG
ytisrevinU of Hawaii at Manoa, ,ululonoH ,sci State ytisrevinU of New York at Al-
Hawaii 22869 ,ynab Albany, New York 22221
xii CONTRIBUTORS TO VOLUME 224
YRREJ L. MOTHGILS (19), Molecular ygoloiB ygolo and Department of ,ygoloiB -revinU
,tinU The Upjohn Company, ,oozamalaK sity of Michigan, Ann Arbor, Michigan
Michigan 49007 90184
SEMAJ F. SMITH (2), Department of ,ygoloiB NEERON TffROSS (9), noitavresnoC Analyti-
Boise State ,ytisrevinU Boise, Idaho 52738 lac Laboratory, Smithsonian Institution,
ARDNAS J. SMITH-GILL (36), Laboratory of ,notgnihsaW D.C. 20560
,sciteneG National Cancer Institute, Na- MARC H. V. VAN LETROMNEGER (10), La-
tional Institutes of Health, Bethesda, eriotarob d'Immunochimie, Institute de
Maryland 29802 eigoloiB Moleculaire et ,erialulleC CNRS,
MARK S. REGNIRPS (17), tnemtrapeD of -iB 48076 ,gruobsartS France
,ygolo University of ,ainrofilaC ,edisreviR CARL WETTER (10), Department of ,ynatoB
ainrofilaC 12529 ytisrevinU of ,nekcifrbraaS ,nekcarbraaS
DAVID STAHL (27), Departments of -ireteV ynamreG
nary ygoloibohtaP and ,ygoloiborciM -inU WARD C. WHEELER (4), Department of In-
versity of lllinois, ,anabrU Illinois 10816 ,setarbetrev American Museum of Natural
LINDA SOLPOHTATS (9), noitavresnoC Ana- ,yrotsiH New York, New York 42001
lytical Laboratory, Smithsonian Institu- HOLLY A. WICHMAN (22), Department of
,noit ,notgnihsaW D.C. 20560 lacigoloiB Sciences, University of ,ohadI
DIANA B. STEIN (12), Department of -goloiB ,wocsoM Idaho 34838
ical Sciences, Mount Holyoke ,egelloC ANNIE K. WILLIAMS (14), Department of
South Hadley, Massachusetts 57010 ,ygolomotnE American Museum of Natu-
HTEB-ORAC TRAWETS (43), Department of lar History, New York, New York 42001
lacigoloiB Sciences, State University of JOHN G. K. WILLIAMS (21), Pioneer Hi-bred
New York at Albany, Albany, New York ,lanoitanretnI Inc., Johnston, Iowa 13105
22221 ALLAN C. ~NOSLIW 1( ,1 42), Division of
YOUNGnAE HUS (1), Natural Products Re- Biochemistry and Molecular ,ygoloiB -inU
search Institute, Seoul National -revinU versity of ,ainrofilaC Berkeley, ainrofilaC
sity, ,064-0111uoeS South aeroK 02749
HTENN~K J. AMSTYS (2), Department of -oB EMEARG WISTOW (41), Section no -uceloM
tany, University of Wisconsin, Madison, ral Structure and Function, National Eye
nisnocsiW 60735 Institute, National Institutes of Health,
W. I~LLEY THOMAS (28, 30), Division of ,adsehteB Maryland 29802
Biochemistry and Molecular ,ygoloiB -inU HTEBAZILE A. ZIMMER (39), Laboratory of
versity of ,ainrofilaC Berkeley, ainrofilaC raluceloM Systematics, National Museum
02749 of Natural History, Smithsonian Institu-
ALLICSIRP K. TUCKER (37), Museum of Zo- ,noit ,notgnihsaW D.C. 20560
3
1 EQUIPPING AND ORGANIZING LABORATORIES
1 Equipping and Organizing Comparative Molecular
Genetics Laboratories
By YOUNGBAE SUH, JUDITH A. BLAKE, and MICHAEL J. BRAUN
Introduction
Rapid advances in biochemical and molecular genetic technology are
revolutionizing many fields of biological inquiry. Nowhere is the effect of
these new tools more keenly felt than in evolutionary biology? For here,
the new technology is not merely changing the course of research within a
discipline; it is merging what were previously unrelated fields. Thus, mod-
ern systematists must now concern themselves with such unlikely topics as
protein structure and function, while molecular geneticists must consider
their data in the fight of evolutionary theory. This melding process can be
especially difficult for biologists without extensive training in biochemistry
or molecular genetics who nonetheless find molecular data sufficiently
compelling to try to use the techniques themselves.
Our purpose in this chapter is to give guidance to the newcomer on how
to organize and equip a laboratory effectively for comparative molecular
research. General articles on laboratory setup can be found in molecular
methodology publications. 3,2 In molecular evolutionary studies, however,
the focus on comparative analysis dictates special emphasis on streamlin-
ing repetitive procedures, adapting techniques for a variety of organisms,
and managing many samples efficiently. We shall endeavor to orient re-
searchers to the general considerations involved in planning a laboratory
for molecular evolutionary studies. We briefly describe the techniques
available and criteria for choosing among them. We suggest rationales for
selecting equipment and strategies for the physical organization of the
laboratory. Throughout, we highlight possible problems and solutions.
Setting Goals
Starting a molecular laboratory requires both consideration of the ulti-
mate questions being addressed by the research and evaluation of the
1 M. Clegg, J. Felsenstein, W. Fitch, M. Goodman, D. ,~.illi4I M. Riley, F. Ruddle, D. Sankoff,
P. Arzberger, M. Courtney, P. Harriman, C. Lynch, J. Plesset, M. Weiss, and T. Yates, Mol.
Phylogenet. Evol. 1, 84 (1992).
2 D. D. Blumberg, this series, Vol. 152, p. 3.
3 C. Orrego, in "PCR Protocols" (M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White,
eds.), p. 447. Academic Press, New York, 1990.
Copydsht © 1993 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 224 All rishts of reproduction in any form reserved.
4 PRACTICAL SEUSSI 1
current research environment available to the researcher. The rapid prolif-
eration and simplification of molecular techniques presents investigators
with what at times may seem a bewildering array of possible approaches to
problems. Yet, from the viewpoint of many evolutionary biologists, these
procedures are still expensive, laborious, and time-consuming. Thus, it is
important at the outset to assess the resources and support available for the
contemplated research, and to pay attention to choosing techniques that
are well suited to the problems at hand.
GOALS
TECHNIQUES
(Ass ss x,s-n,. . sou.o sJ
RESOURCES
.1 LACISYHP I .1 LANRETNI I &"
.2 LAICNANIF .2 LANRETXE
.3 NAMUH .3 DERAHS
PLAN LABORATORY FACILITY ~ ........ PLANNING
I
( PERF(~Ivl HCRAESER ~ RESEARCH
FIG. .1 Flowchart for laboratory planning. The five major stages in planning a laboratory
facility are listed to the fight. Arrows in the chart indicate the direction of progress through
stages and how information gleaned at one stage can cause reconsideration of decisions made
at a previous stage.
1 EQUIPPING AND ORGANIZING LABORATORIES 5
The following issues will merit consideration: (1) What frequency and
intensity of laboratory work is expected? Some scientists need to obtain
molecular data on one or a few specific questions; for others it will be an
ongoing focus of research. (2) What technique or techniques will be used?
Success often hinges on choosing a technique well suited to the research
problem. The scope of the laboratory operation, in turn, will increase with
the number and complexity of techniques to be implemented. (3) What
resources already exist? What resources are obtainable? Given the cost and
complexity of molecular research, it is crucial to assess the physical, tech-
nical, and human resources of the home institution and the local environ-
ment, and to be realistic about opportunities for obtaining new resources.
(4) Can resources be shared? Virtually all molecular investigators share
some laboratory resources. The extent of sharing may range from a few
pieces of large equipment in a traditional department comprising many
single-investigator laboratories to sharing of a single, centralized molecular
facility among several investigators. Collaboration among research groups
can provide the means to address scientific questions without setting up
multiple independent research units.
Careful consideration of these basic issues at the outset will lead to
more effective planning and a cost-e$cient facility better designed to meet
the needs of the individual and the institution. A flowchart (Fig. )1 helps
illustrate the interrelationships of these issues and the feedback effects they
may have on one another.
Choosing Techniques
The physical organization of a laboratory will be dictated by the tech-
niques to be implemented there. Thus, it is essential to decide what tech-
niques will be used immediately and what techniques might be attempted
at a later date. Six major comparative molecular techniques are in com-
mon use today in evolutionary research. These are isozyme dectrophore-
sis, comparative immunological methods especially microcomplement
fixation (MC'F), DNA-DNA hybridization, restriction enzyme analysis,
random amplification of polymorphic DNA (RAPD), and DNA sequenc-
ing. Two other methods, molecular cloning and the polymerase chain
reaction (PCR), are not inherently comparative, but they provide the
foundation on which a number of DNA techniques are based. We first
consider the major factors involved in choosing among techniques, then
discuss the advantages and disadvantages of each. Table I summarizes
information presented below that will be helpful in selecting techniques.
6 PRACTICAL ISSUES 1
TABLE I
CONSIDERATIONS FOR CHOOSING TECHNIQUES a
lamitpO
ecnegrevid emiT dna
euqinhceT range Complexity Cost labor Nature of samples ytilibarapmoC
emyzosI electro- Lo/Med oL oL oL elpitluM seussit dooG
siserohp
tnemelpmocorciM deM iH deM Med/Hi Purified nietorp riaF
noitaxif
DNA-DNA -yh deM Hi deM Med/Hi Large seititnauq of riaF
noitazidirb AND
noitcirtseR emyzne oL deM Hi Med/Hi WMH AND dooG
sisylana
AND sequencing Lo/Med/Hi Hi Hi Hi ynA deifirup AND Excellent
modnaR -acifilpma oL deM deM oL WMH AND riaF
noit of -romylop
cihp AND
esaremyloP niahc .A.N deM deM oL ynA deifirup AND .A.N
noitcaer
raluceloM cloning N.A. Hi Hi Hi ynA deifirup AND .A.N
,.A.N toN ,elbacilppa ecnis euqinhcet si ton a evitarapmoc ,dohtem but smrof eht sisab rof ;srehto
,WMH hgih ralucelom ;thgiew ,oL ;wol ,deM ;muidem ,iH .hgih
gnihctaM gnivloseR rewoP of seuqinhceT ot citeneG ecnegreviD of
smsinagrO
Most research problems in molecular evolutionary genetics ultimately
involve questions of relationship. However, the degree of relationship may
vary dramatically, from comparisons of related individuals within a popu-
lation to comparisons of taxa that diverged early in the history of life on
Earth. There is a general correlation between degree of relationship among
organisms and the level of genetic similarity among their genomes. Again,
the level of similarity can vary widely: the genomes of clonally reproducing
organisms may be nearly 100% identical, whereas those of distantly diver-
gent taxa may retain sequence similarity only in a few, highly conserved
genes. The level of genetic similarity among the genes or genomes to be
compared is the primary determinant of the applicability of the various
comparative molecular techniques. Each technique is best suited to a
particular range of divergences among the organisms being compared.
Each technique probes genetic questions at different levels of resolution,
from direct DNA sequence comparisons to inferred amino acid sequence
variation. Some techniques, such as DNA sequencing, can be adapted to a
broad range of divergences; others are not so versatile. Failure to appreciate
1 EQUIPPING AND ORGANIZING LABORATORIES 7
the relationship between gene or genome divergence and resolving power
of a technique can lead to a data set that is largely irrelevant to the original
research focus.
ytixelpmoC
To use some methods successfully requires more background and ex-
perience in biochemistry than others. Most comparative techniques in-
volve repetitive procedures that seem deceptively simple to learn when all
is working well. When a technique fails to work properly, however, consid-
erable expertise may be required to discover the problem and get the
research back on track.
tsoC
It is convenient to think of costs as falling into three categories, namely,
overhead costs of setting up and maintaining the laboratory, consumables
cost of the supplies actually used up in a particular project, and personnel
costs. At present, initial overhead costs might range from $20,000 or less
for a laboratory concentrating on isozyme surveys to several hundred
thousand dollars for a laboratory fully equipped to implement a wide range
of techniques. Setup costs will vary dramatically depending on preexisting
resources and physical location, so it is important to explore the local
environment of the proposed laboratory (see section on assessing environ-
ment and resources below). The annual cost of consumable supplies will
range from a few thousand dollars per active researcher for isozyme work
to $10,000-$15,000 per person for the more expensive techniques. At
many university laboratories, personnel costs may not be a major factor if
salaries and stipends come from sources other than research funds. At
other institutions, however, where all expenditures come from a common
pool, personnel costs are likely to be an important budget item.
Time and Labor
The major comparative techniques differ substantially in the amount of
time and effort they require. Some labor-intensive techniques are more
appropriate when relatively small numbers of comparisons are to be made;
other techniques can be readily applied to large numbers of samples.
Nature of msinagrO ro~dna selpmaS
The techniques differ in the amount and type of sample required. The
physical size of the organism may limit the amount of tissue that can be
obtained from each individual, and may make some techniques difficult or
8 LACITCARP msuEs 1
impossible to use. Some techniques work best with certain kinds of tissue.
Other techniques (e.g., isozyme electrophoresis) benefit from the availabil-
ity of several tissue types. Most techniques work best with fresh or frozen
material, but some can be used with samples that are thousands of years
old.
Comparability across Studies
The ease with which a data set can be compared to other studies is an
important consideration. DNA sequencing provides the greatest advan-
tages in this regard. Because the method involves the absolute determina-
tion of nucleotide sequence, the data can be compared to other sequences
determined completely independently. Some data derived from other
techniques can be compared in a general way (e.g., isozyme genetic dis-
tance, sequence divergence estimated from restriction enzyme analysis),
whereas other comparisons are best made side by side in the same labora-
tory (e.g., establishing homology of isozyme alleles or restriction fragment
bands).
Techniques: Pros and Cons
Isozyme siserohportcelE
The examination of proteins by electrophoresis has been the single
most popular technique in molecular evolutionary genetics. It can provide
useful information for studies at the population and species level. 6-4 For
some taxa where genetic distances tend to be low, such as birds, the useful
range of isozyme electrophoresis extends to the genus and even family
level. It is the least expensive technique, both in terms of overhead and cost
of consumables. Large sample sizes can be handled readily, and the techni-
cal demands are relatively minimal. Electrophoresis and staining condi-
tions are often similar throughout major groups of organisms, although
some optimization is required for most studies. 7-4 In addition, gene ex-
pression varies by tissue, so having a variety of tissue types available for
study increases the number of genes that can be assayed. Although data
from separate studies cannot be directly combined, the large body of
comparative data available in the literature often provides a helpful back-
ground for interpretation of isozyme data.
4 C. .J Bult and Y.-T. Kiang, siht volume .6
s .j ,naidivaD-nottirB siht volume .7
R. W. Murphy, .J W. ,setiS ,.rJ D. G. Buth, and C. H. ,relfnaH in "Molecular "scitametsyS
(D. M. silliH and C. Moritz, ,).sde .p .54 ,reuaniS Sunderland, ,sttesuhcassaM 1990.
D. Goldman and .S .J ,neirB'O siht volume .8