Kasbekar, Durgadas P.

English

Publications of the IAS Fellows

Commentary
Successful beyond expectation: David Perkins’s research with 
chromosome rearrangements in Neurospora
The Thomas Hunt Morgan Medal is awarded by the Genetics Society of America to recognize a lifetime 
contribution to genetics. The fi rst awardees were Barbara McClintock and Marcus Rhoades, in 1981.  In 
1994 this Medal was awarded to David D. Perkins of Stanford University.  By then David had shown that 
many different genetic properties of eukaryotes, including recombination, chromosome rearrangements 
and meiotic drive; can be studied using Neurospora as a model.  David had also made compilations of 
all known Neurospora mutants, chromosome rearrangements, wild and multiply marked strains, linkage 
maps, methods, publications, and (more recently) useful websites.  He was the prime mover in instituting 
an annual Newsletter and establishing the Fungal Genetics Stock Center (www.fgsc.net).  In the Report 
of the Committee on Honors and Awards, David R. Stadler wrote  – “For those of us who work on the 
genetics of Neurospora, a day seldom passes that we don’t give thanks to a generous Providence for David 
Perkins.  So many times he has made life’s path a little easier for us” (Genetics 137:s12-s13, 1994).  
David Perkins is best known for developing a simple and elegant method to detect and distinguish 
different kinds of chromosome rearrangements (inversions, intrachromosomal transpositions, reciprocal 
translocations and insertional and quasiterminal translocations).  In a single-author tour-de-force paper that 
he dedicated to Curt Stern, his undergraduate mentor at the University of Rochester, David diagnosed >135 
rearrangements by mere visual inspection of black (viable, non-defi ciency) and white (inviable, defi ciency) 
ascospores in unordered tetrads (actually, octads) from Rearrangement x Normal crosses (Perkins 1974). 
Five classes of tetrads are possible; 8B:0W; 6B: 2W; 4B:4W; 2B:6W and 0B:8W.   Isosequential crosses 
produce 90-95% 8:0 tetrads but a Reciprocal Translocation (RT) x Normal cross produces equal numbers 
of 8:0’s and 0:8’s.  The former result from ‘alternate’ segregation (which generates the parental types) 
whereas the latter are from “adjacent 1” segregation (which generates duplication/defi ciency types).  Any 
4:4’s signal a crossover between the centromere and the translocation breakpoint, and no 6:2’s and 2:
6’s are expected.  In Insertional Translocations (IT), a segment of one linkage group is translocated into 
another linkage group.  If the ‘recipient’ chromosome from the IT parent of an IT x N cross (N = “normal 
sequence”) segregates with the N homolog of the ‘donor’ chromosome, then the resulting segregant is 
duplicated (Dp) for the translocated segment and only the complementary ‘defi ciency’ segregants are 
inviable.  Therefore in IT x N crosses 8:0’s = 4:4’s, whereas 6:2’s signal interstitial crossover, and 0:8’s 
and 2:6’s are rare.  Thus the frequency distribution of the different tetrad classes is a characteristic of the 
kind of rearrangement and provides information as to the position of breaks relative to the centromere.  
None other than Barbara McClintock encouraged David to undertake this study, and in gratitude David 
exulted “The method proved successful beyond expectation …” (Perkins, 1992).  David’s 159 page 
review (Perkins 1997) describes more than 300 rearrangements that he analyzed over a 30 year period of 
work, and also discusses the pitfalls in identifying and diagnosing rearrangements.  It remains to this day 
the defi nitive reference work on the subject.  
David had a rare knack for doing crosses with carefully chosen markers to solve the structure of 
rearranged chromosomes, supplemented, when necessary, with cytological and molecular studies.  Not 
surprisingly, his diligence led him to make novel and unexpected discoveries.  For example, in his effort 
to determine the orientation of the transposed segment in the intrachromosomal transposition Tp(IR> 
IL)T54M94, David wanted to make a Tp x Tp cross heterozygous for markers both inside and outside 
the transposed segment.  He had with “… great diffi culty …” (by screening for a rare double crossover) 
inserted one marker (cyh-1) into the transposed segment, but needed two to obtain the needed evidence.  
My guess is at this stage a lesser scientist would throw in the towel (- why determine orientation of 
http://www.ias.ac.in/jbiosci J. Biosci. 32(2), March 2007, 191–195, © Indian Academy of Sciences    191
Keywords. Meiotic silencing; quelling; repeat-induced point mutation; Spore killers
Commentary192
J. Biosci. 32(2), March 2007
an intrachromosomal transposition anyway?).  Not David, he decided to insert an al-2 marker, but via 
repeat-induced point mutation (RIP); a genome defense process that occurs in the premeiosis of a sexual 
cross and effi ciently mutates duplicated DNA sequences (presumably, to destroy transposable and other 
parasitic DNA elements).  To duplicate the al-2 gene sequence David and colleagues transformed the Tp 
strain with an as yet unpublished al-2 clone (from T L Schmidhauser).  To their surprise, many primary 
transformants showed an unstable albino phenotype; Perkins and colleagues had inadvertently (and 
independently) stumbled upon ‘quelling’, an RNAi-mediated gene-silencing phenomenon that silences 
transgenes and their endogenous homologues.  Happily, they could also generate the al-2 mutation and 
used it to establish that the transposed segment was “… inserted near the centromere in the opposite arm, 
in inverted order” (Perkins et al 1995).
The 1995 paper also illustrates the elegant use made by David and colleagues of the meiotic drive 
element Spore killer-2 (Sk-2) to map the insertion site of the transposed segment with respect to the 
centromere.  Spore killers –2 and –3 (Sk-2 and Sk-3) are gene complexes (haplotypes), tightly linked to 
the centromere of linkage group III, that David and Barbara Turner identifi ed in a subset of Neurospora 
intermedia strains David isolated from Borneo (Brunei), Java and Papua New Guinea (Turner, 2001).  No 
killers were found in N. crassa, but the N. intermedia  Sk  factors are fully functional when introgressed 
into N. crassa and N. tetrasperma  (Raju and Perkins, 1991).  Sk x wild type crosses produce 4:4 tetrads; 
the black (viable) ascospores are ones that inherit Sk, whereas the white (inviable) ascospores are those 
that inherit the wild type linkage group III (fi gure 1).  David and colleagues had earlier shown that Sk-2 
and Sk-3 are useful for obtaining information from unordered asci that otherwise would require ordered 
asci  (Perkins et al 1986).  Since the Sk complexes are centromere-linked, they segregate at the fi rst meiotic 
division.  Ascospores are inviable in the half-tetrad that is devoid of Sk.  Thus a marker gene is known to 
have segregated at the fi rst division if the same allele is present in both ascospore pairs of the surviving 
half-ascus.  If the ascospore pairs of the surviving half-ascus contain unlike alleles then the marker must 
have segregated at the second meiotic division, i.e., it must have undergone recombination with respect 
to its centromere.  As the 1986 paper put it, “…(effectively), physically unordered asci are changed into 
ordered tetrads making it unnecessary to dissect ascospores from intact eight-spored asci.  Availability 
of this easy method is not expected to produce any revolutionary new types of information.  Rather, its 
signifi cance is likely to be that the method will make it practical to obtain useful information that would 
previously have seemed too laborious to seek.”
Figure 1. A rosette of asynchronous asci from the cross Sk x wild type.  Each mature ascus shows four large black 
(viable) ascospores and four small aborted ascospores.  All the viable ascospores are genotypically Sk.  From Raju 
1980, Eur. J. Cell Biol. 23 208–223, reproduced here with permission from the author.
Commentary 193
J. Biosci. 32(2), March 2007
David and colleagues reported many other interesting results on Spore killers.  Turner and Perkins 
(1979) showed that no killing occurs in homozygous Sk-2  x Sk-2 and Sk-3 x Sk-3 crosses and that all 
ascospores are killed in Sk-2 x Sk-3 intercrosses.  And in both Neurospora intermedia and N. crassa they 
identifi ed resistant strains that neither kill nor are killed.  But my favourite was the demonstration of a 
Spore killer-like programmed ascospore death in the homothallic species Coniochaeta tetraspora (Raju 
and Perkins 2000).  In homothallic species the mycelium from a single, initially uninucleate ascospore 
is capable of completing the sexual cycle.  Thus all the nuclei participating in a cross are identical.  
Nevertheless, of the eight ascospores cut out in each ascus, four die and only the other four are viable, just 
as in a Neurospora Sk-2 x WT cross.  What maintains this system of apparently willful waste?
David crafted rearranged chromosomes into versatile, Swiss Army Knife-like tools.  For example, to 
increase the effi ciency of linkage detection he marked three reciprocal translocations with “easy to score” 
markers and brought them together in the alcoy linkage-tester strains (T(I;II)4637 al-1; T(IV;V)R2355, 
cot-1; T(III;VI)1, ylo-1; csp-2).  Except for the marker csp-2, which marks linkage group VII, each of the 
other markers (al-1, cot-1 and ylo-1) tags two linkage groups (Perkins et al 1969).  If a point mutation in 
a mutant x alcoy cross shows linkage to one of the three alcoy markers, one only needs to do a follow-up 
cross with a normal-sequence tester to determine which of the alternatives is correct.  A mutation that 
shows linkage to two markers very likely signals a new translocation.
Dp progeny from crosses of the type IT x N, RT x overlapping RT, or Inversion x overlapping Inversion, 
were used for a variety of purposes, including establishing correct gene order among closely linked loci 
(Perkins 1986), localizing vegetative incompatibility loci (Perkins 1975) and to detect escape from 
Figure 2. David Perkins with Prime Minister Indira Gandhi and M S Swaminathan at the International Genetics 
Congress in New Delhi, December 1983. (Photo from Susan  Perkins.)
Commentary194
J. Biosci. 32(2), March 2007
vegetative incompatibility via chromosome breakage (Perkins et al 1972). Dp segregants are recognizable 
by the barrenness of Dp x N crosses.  Barren crosses make perithecia but produce very few ascospores.  
The barrenness of Dp x N crosses is now known to be due to the silencing of duplication-borne genes 
by another RNAi-mediated gene-silencing process, which silences genes unpaired in meiosis as well as 
their homologues.  Robert Metzenberg and colleagues, including David’s associate N B Raju, discovered 
meiotic silencing and also isolated semi-dominant suppressors of this process (Shiu et al 2001).  Much 
of this work was done during Metzenberg’s stay in David’s lab in the late 90’s and early 00’s.  The 
suppressors of meiotic silencing have now made it easier to obtain large numbers of progeny from the 
otherwise barren Dp x N crosses.
Much of my own research was sparked by David’s demonstration of RIP in large duplications (Perkins 
et al 1997).  This prompted us to test, and thus discover, that a small gene-sized (~1-3 kb) duplication 
escapes RIP when a large (>300 kb) duplication is present in the cross (Bhat and Kasbekar 2001), possibly 
because the large duplication titrates out the RIP machinery.  Armed with the knowledge that one can screen 
for genotypes that confer a dominant RIP suppressor phenotype, we screened for this phenotype among 
all the >400 wild-isolated strains of N. crassa obtainable from the Fungal Genetics Stock Center, most 
were collected by David on trips made around the tropical world (Turner et al 2001).  Seven wild isolates 
(including one naturally occurring duplication strain) had the dominant RIP suppressor phenotype (Bhat 
et al 2003; Vyas et al 2006).  David encouraged me to use Neurospora tetrasperma, a pseudohomothallic 
species, to screen for recessive mutants defective in RIP and meiotic silencing.  Many years previously 
Francis Ryan took David, then a graduate student at Columbia University, to the New York Botanical 
Garden up in the Bronx, to visit B. O. Dodge, the discoverer of both N. crassa and N. tetrasperma.  N. 
tetrasperma was Dodge’s favorite species and he tried (without success) to get his visitors interested in 
using it.  Dodge even offered all his slides related to tetrasperma cytology to anyone who would use them.  
David confi ded to me that Dodge would have been pleased to see what we were doing (Bhat et al 2004).  
David also contributed more directly to our articles. I treasure the detailed handwritten comments and 
suggestions that he made on our manuscripts.  These were sent both by fax (because he knew we were 
impatient) and by regular mail.  Often he redrew our fi gures to make them more understandable to a larger 
audience and once even re-wrote the abstract! 
David Perkins passed away on January 2, 2007, four months short of his 88th birthday. His wife 
Dorothy Newmeyer Perkins (Dot) passed away peacefully on January 6, and joined David Perkins. Dot 
was 84 years old. They are survived by their daughter Susan and her husband John.
Acknowledgements
Writing this tribute would not have been possible without the reassurance and critical inputs I received 
from Namboori Raju, Sharat Chandra, Ramesh Maheshwari, K Dharmalingam, P Maruthi Mohan,
D Balasubramanian and Imran Siddiqi.
References
Bhat A and Kasbekar DP  2001  Escape from repeat-induced point mutation of a gene-sized duplication in Neurospora 
crassa crosses that are heterozygous for a larger chromosome segment duplication; Genetics 157 1581–1590 
Bhat A, Noubissi F K, Vyas M and Kasbekar D P 2003  Genetic analysis of wild-isolated Neurospora crassa strains 
identifi ed as dominant suppressors of repeat-induced point mutation (RIP);  Genetics 164 947–961
Bhat A, Tamuli R and Kasbekar D P 2004 Genetic transformation of Neurospora tetrasperma, demonstration of 
repeat-induced point mutation (RIP) in self-crosses, and a screen for recessive RIP-defective mutants; Genetics 
167 1155–1164
Perkins D D 1974 The manifestation of chromosome rearrangements in unordered asci of Neurospora; Genetics 77 
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Commentary 195
J. Biosci. 32(2), March 2007
Perkins D D 1992  Neurospora chromosomes: in The dynamic genome: Barbara McClintock’s ideas in the century of 
genetics (eds) N Federoff and D Botstein (New York: Cold Spring Harbor Laboratory Press) pp 33–44
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Neurospora; Chromosoma 104 260–273  
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segmental duplications of Neurospora; Genetics 147 125–136   
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Neurospora tetrasperma; Genetics 129 25–37  
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containing it; Genetics 93 587–606  
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DURGADAS P KASBEKAR
Centre for Cellular and Molecular Biology,
Hyderabad 500 007, India
(Email, kas@ccmb.res.in)
ePublication: 31 January 2007


Successful beyond expectation: David Perkins's research with chromosome rearrangements in Neurospora

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Successful beyond expectation: David Perkins's research with chromosome rearrangements in Neurospora

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