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ORN-peare
CONF-734-2
NOV 1 3 1904
MASTER
INTERACTION BETWEEN NONALLLIC MUTASIONS IN NEUROSPORA*
Frederick J. de Serres
Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee
---LEGAL NOTICE

Tv. raport memanda Mi Gorenmen moun wont. Mother the United
Y ., nor the Canalu , MY NO ponudu kthy
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nxy, topelmen, or w hen of de heridoneum thanh tmpor, or what the one
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A.
A m ury luwon wa rompact both of, a hor damage toute from the
in un lortuidon, mani, wehed, men untowed to worsport,
Al w te the show, per intet an all of the Coronto " included w h e
payne or contractor de Cocaminet, upto w wart contractor, to the one dat
ml employee or contractor at the Court or empien och cirector mapama,
dinseminatre, er providen verran to, aky llormatien perusomt to bilo employment or metroet
to the Commtuulon, or wo employ tumot so much contractor.
*Research sponsored by the V. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
Running head:
Send proof to: Dr. Frederick J. de Serres
Biology Division
Oak Ridge National Laboratory
P. O. Box Y
Oak Ridge, Tennessee
I
*
*
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.
.
One of the most intriguing areas for genetio analysis is the study of
the control or gene function. At one time it was generally abowned that the
action of genes producing functional proteins was autonomous, but many moont
Investigations indicate that the primary action of structural genes 16 Bubject
to various mechanisms of control. In the prokaryotes, studies on genes
affecting histidine biosynthesis in Sulmonella (Hartman, Loper and Serman 1960,
Ames and Hartman 1962, 1963) and on the B-galactosidase system in Escherichia
coll (Jacob and Monod 1961) have demonstrated a mechanism for the coordinate
control of the action of linked genes affecting the same biosynthetic pathway
by means of operator genes. Each of these regions constitutes a unit of
coordinated transcription termed an operon (Jacob and Monod 1961). That such
control mechanisms are not limited to the prokaryotes has been shown by Giles
and his associates (Giles 1964, Ahmed, Case and Giles 1964) who have demonstrated
the existence of an operon in the eukaryote Neuirospora. These investigators
have shown that the studies of Webber (1959, 19642, b) and Ahmed (Ahmed et al.
1964) can be interpreted in terms of an operon and they have proposed that the
hist-3 region consists of an operator gene and at least three genes controlling
different steps in 'histidine biosynthesis. Other types of control mechanisms
have undoubtedly evolved for the regulation of gene action with the organization
of the DNA into chromosomes of more complex structure and function, and studies
on other eukaryotes have provided evidence for more complex types of interaction.
Work done on the relations between genes in maize has shown that the action
(and mutability) of a structural gene appears to be determined by the presence
of controlling elements at or near the gene locus (McClintock 1956). In
addition, in Drosophila complex effects on the function of genes have been
observed when their associations with neighboring genes 1s changed as a
consequence of certain types of X-ray-induced chromosome rearrangemente
(review Lewis 1990). This latter category of effoots has been referred to
as position effect mutations and they may be either of the variegated or
stable type. In brief, thebe examplos show that the songs are not
autonomous units and that their primary activity can probably be regulated
by various types of control mechanioms in all organioms.
One of the most intriguing üypes of position effects in Drosophila are
those with "spreading effects" that result in the variegation of several genes
in the vicinity of the point of chromosome rearrangement. It has been shown
in Drosophila that variegated-type position effects and associated with
euchromatin and heterochromatin rearrangement, but the precise mechanisms
responsible for the spreading effects associated with certain chromosome
rearrangements are unknown. Although many of the experiments in this general
area are 30 to 40 years old, investigation of the mechanisms responsible for
position effects 18 still one of the relatively unexplored areas of genetics.
The rapid technical advances in the use of some of the "microorganisms" among
the eukaryotes for genetic analysis in recent years makes possible novel
approaches to these fundamental problems. Since the DNA in the green algae
and the fungi is organized into chromosomes in a manner similar to if not'
identical with that found in higher organisms, it should be possible these
problems can be investigated at the biochemical level.
In Neurospora, nonallelic mutations show full complementation and .
heterokaryons between nonallelic biochemical markers grown at wild type rate.
Departures from this generality can usually be attributed to the unsuspected
heterozygosity for factors affecting either heterokaryon formation or
maintenance (Garnjobst 1953, 1955, Holloway 1955, de Serres 1962, Pittenger
and Brawner 1961). Mutations resulting in position effects, studied 80
extonsively in Drosophild, are unknown in microorganisms. It is pogodble
that this is due to the simple fact that such mutations are haploid or
homokaryotio lethal. In Neurospora the only mutations that have been studied
extensively are those that can grow as homokaryons on supplemented minimal
medium or completo medium. Such mutations have been classified as "reparable
mutations" (Atwood and Mikai 1953), and they are reparable in the sense that
the gonetio alteration leads to a requirement that can be satisfied by
(usually a single) biochemical supplementation of the basal minimal medium.
Irreparable mutations are also known and these behave as recessive lethals
even un complete medium (Atwood and Mukai 1953).
Recently the development of a new procedure for forward-mutation experiments
in Neurospora (de Serres and Osterbind 1962, Brockman and de Serres 1963) makes
1t possible to induce, maintain and analyze the same spectrum of senetic
alterations at specific loci in this organism as 18 found at specific loci
in diploid organisme. This procedure makes use of a 2-component heterokaryon
and both homkaryotic viable (reparable mutations) and homokaryotic lethal
(irreparable mutations) have been recovered (de Serres and Osterbind 1962).
Other techniques have been developed for the identification of those mutations
with effects more complex than the functional inactivation of a single genetic
locus. Genetic analyses of the reparable and irreparable mutations induced at
closely linked loci in the ad-3 region of Neurospora have provided evidence
for position effects in Neurospora and a means for the investigation of this
phenomenon in microorganisms. The investigations reported here represent the
first attempts towards the identification of mutations with spreading effects
and the development of techniques and approaches for more critical genetic
analysis.
Mutant Isolation
The mutations for this anayois were induced at two different but closely
linked loci in the ad-3 rogion of Neuros pora designated ad-3A and ad-3B
(de Serras 1956). Mutatiuns at either of these loci are phenotypioally
identical; they accumulate purple pigment in the vacuoles of the mycelium
and have a requirement for adenino. Biochemical analyses have shown that
they affect the same or sequential steps in purine biosynthosis (Lukens and
Buchanan 1959). Each component of the heterokaryon (referred to as a dikaryon)
used in the forward-mutation experiments contains a series of biochemical
markers as shown in Table 1. This dikaryon was designed so that any genetic
alteration that inactivates the ad-3A or ad-3B locus, or both, in component II
in a heterokaryotic conidium, will result in the formation of a reddish-purple
(T-1)
colony in adenine-supplemented medium. All other heterokaryotic conidia form
colorless colonies and because of the difference in the pigmentation between
mutant and nonmutant colonies those colonies containing 21-3 mutations can
be recovered by a direct method (de Serres and Kølmark 1958, Brockman and de Serres
1963). With this method the entire population of treated conidia are incubated
and each conidium surviving mutagenic treatment forms a microcolony about
2 ma in diameter. With an average of 10° colonies per incubation flask and
forward-mutation rates ranging from 30 to 300 per 10° survivors (de Serres
19640) recovery of large numbers of mutants after treatment with specific
mutagens is readily accomplished.
Characterization of ad-3 mutations
Genetic analysis of the mutations induced in a dikaryon after X-ray
treatment showed that these mutations can be subdivided into two main classes:
reparablo mutations (ad-3") that will grow as homokaryons on adenine
supplemented medium and irreparable mutations (ad-zek) that will not grow
as homokaryone even on complete medium (de Serres 1964a). The reparable
mutations are always of genotype ad-3A or ad-3B, but the irreparable mutations
can be oither ad-3A, ad-ZB or ad-3A ad-3B double mutants. Genotypes are
detormined by a series of simple hoterokaryon tests with four different
tester strains of the following genotypes (1) ad-31, (2) ad-3B, (3)
hist-2 nio-2, and (4) ad-2 1nos. Since the markers in each of these four
tester strains are found in component I of the dikaryon, such heterokaryon
tests involve only the reaction of component II of the dikaryon with the
testers.
IR
Interaction between ad-3* and ad-34mutations in forced dikaryons
The interaction between the reparable and irreparable mutations at these
two loci was studied first by combining them in various pairwise combinations
in forced dikaryone and then determining their linear growth rates. This was
accomplished by forcing heterokaryon formation between the dikaryons containing
reparable or irreparable mutations at the ad-3A locus with other dikaryong
containing reparable or irreparable mutations at the ad-3B locus to form,
tetrakaryons (really trikaryons since 2 of the 4 components are identical in
genotype) and then extracting the forced dikaryons with these nonallelic
mutations in pairwise combination as indicated in figure 1. When these
forced dikaryons were isolated from the conidial platings of the trikaryons
and single colony 1solates were transferred to the bottom of an agar blant in
test tubes, it was apparent that there were marked differences in the growth
rates of different mutant combinations. The slow growth of the combinations
of irreparable mutants were particularly slow, whereas those involving
ad-3* + ad-ZIR combinations or ad-Z* + ad-38" combinations appeared to grow
at wild type rate.
It was impossible to obtain forced dikaryons for some of the combinations
of irreparable mutations (de Serres 1964a), and the significance of this
observation will be discussed in a later section.
To obtain more precise data on the growth of these forced dikaryons, che
linear growth rates were determined in horizontal growth tubes (Ryan, Beadle
and Tatum 1943). Heavy suspensions vf conidia from two single colonly isolates
of each dikaryon (about 10° conidia/m2) were used to inoculate duplicate growth
tubes containing pantothenate supplenented medium. Each tube was marked daily
for 7 to 30 days to record the total linear growth made in successive 24-hour
periods. There was considerable variation in the linear growth rates of single
colony 160lates from some of the dikaryons. Because the slower growth rates
of some of the single colony isolates can probably be attributed to markedly
skewed nuclear ratios (or more stringent requirements for nuclear ratios
approximating 1:1), a large number was isolated and tested. For the growth
tube experiments only the most rapidly growing single colony isolates of each
combination were used. Average growth rates were determined in the region of
the growth tube distal to that point, either where the growth rate became
constant or where the maximum growth rate was obtained. The linear growth
rates of the pooled data from the analysis of these dikaryons 1s presented
in Table 2. The linear growth rate of strain 74-OR31-16A, component II of
the dikaryon in which these mutations were induced, was 2.83+0.05 in the
same experiment.
The linear growth rates of dikaryons involving ad-3? + ad-zIR combinations
can be best compared with that of the dikaryon between the two reparable mutants
(1-2)
11-0 + 1-1 used as a control. Whereas the majority of these dikaryons grow
as rapidly as 11-0 + 1-2, the dikaryone involving the ad-3A+* mutants 13-0,
122-0, and the ad-3B+mutants 10-1, 121-1 and 123-1 show significant
reductions in lineer growth rate.
The most striking feature of these data 18 the marked reduction in linear
IR
growth rate found with all of the combinations of irreparable mutants as
compared with that of the two reparable mivants 11-0 + 1-1. There 18 almost
a 10-fold difference between the growth rate of this combination and the
growth rate of dikaryon involving 122-0 + 121-1! . . .
The nomal growth of dikaryong involving an ad-3A and an ad-3B mutant
should depend on the presence of a combined complement of undamaged genetic
material in the ad-3 and immediately adjacent regions. It would appear that
the functional inactivation in ad-z* mutants does not extend beyond the boundaries
of the ad-3A or ad-3B cistron, since forced dikaryons between ad-zmutants
have a normally functioning ad-3A and ad-3B locus and grow at wild type rate.
Any spreading effects of the genetic alterations producing ad-ZA-* or ad-3BLK
mutations however would affect the activity of loci in the region in between
the ad-3A and ad-3B locus in addition to loci in the regions beyond both to
the right and to the left. Furthermore the growth rates of various combinations
of reparable and irreparable mutations in pairwise combinations should provide
some measure of any reduction in functional activity. In some cases wild type
growth rate can be obtained with as little as 3% of the wild type enzyme
activity as shown by Woodward (Woodward, Partridge and Giles 1958) for
revertants of ad-4 mutants. If the same situation applies to the ad-ZA or
the ad-3B locus, then sny reduction in growth rate observed could result
quite drastic alterations in the activity of either of these two loci.
IR
That the wild type ad-3A locus shows less than normal function in certain
ad-3BR mutants and the wild type ad-3B locus shows less than normal function
in certain ad-3Amutants 16 evident from the slower rates of growth of some
CETE E + -corbinstics ia izble 1. Tae consequences
cpreading crfccts are enhanced in various ad-3+* + ad-3IR"conited restiers viuere
a vild type ad-34 locus 18 combined with a wild type ad-3B locus where both
have reduced functional activity so that the dikaryons show more striking -
reductions in growth rate.
The nature of the genetic alterations producing ad-3 and ad-3+* mutants
Mutations showing position effects in Drosophila are associated with
such chromosome rearrangements as inversions and translocations (review Lewis
1950). Such extragenic mutations require chromosome breaks in two different
places in the genome and show 2-hit kinetics after treatment with X rays (review
Giles 1954). Point mutations or intragenic alterations result from molecular
rearrangements or alterations within the gene and should show l-hit kinetics.
An attempt was made to determine the nature of the genetic alterations
producing ad-3 and ad-3-K mutations by an analysis of their induction kinetics
after exposure of the dikaryon shown in Table 1 to X rays (Webber and de Serres
1964). In this analysis the frequency of all ad-3 mutants (or purple colonies)
was found to increase as the 1.36 power of the dose. Genetic analysis of
mutants showed that the ad-3 mutants increase in direct proportion to dose
whereas the ad-3 mutants increase as the square of the dose (Figure 2). (F-2) |
IR
locus some 3.0 map units distal were also sought in this experiment and two
additional classes of ad-za mutants were found, the first of genotype
(ad-3B nic-2)TR and the second (ad-3A ad-3B nic-2)R.
This experiment provides direct evidence that ad-34 matants result
from events, and it may be surmised that they result from such extragenic
alterations as inversions, deletions or translocations. It is of particular
interest that ad-3 mutations are not produced in a comparable experiment.
where the same dikaryon was treated with nitrous acid (Brockman, Barnett and
de Serres, in preparation). Ad-ze mutations appear to result solely from
extragenic mutations and are not produced by an agent that produces. mutations
predominantly by base-pair substitution in NEUTOspora (de Serres 1954b,
de Serres, Broclaman, Barnett and Keimurk, in preparation),
The significance of the apparent homology of certain ad-3Aand ad-3B-R
mutations
As mentioned in a previous section it was not possible to isolate a
forced dikaryon homozygous for the markers cot, al-2, pan-2 from the trikaryons
between certain ad-3A+ ad-3B-* combinations: 13-0 + 10-1, 13-0 + 121-1
and 122-0 + 10-1. After several different experimental approaches failed to
yield forced dikaryons between these combinations, we concluded that they must
contain homologous or overlapping areas of functional inactivation (de Serres
1964a). The simplest assumption was that the extensive functional inactivation
associated with these mutations resulted from genetic damage located in the
region between these two loci, so that these mutants have been represeated as
overlapping on a complementation map of the ad-3 region (Figure 3).
The kinetic analysis of the Induction of ad-3? and ad-3IR mutations
with X rays indicates that ad-3* mutants are not converted to ad-3-R mutants
with increasing X ray exposure but rather that ad-zmutants each result
from two hits and that each one of these hits is not detectable as an ad-3
(F-3)
mutation by itself.
There we have assumed that they occur outside of the
boundaries of either the ad-3A or ad-3B locus. The implication of the homology
between certain nonallelic ad-3-* mutations is that they have genetic damage
in common located outside of the ad-3A or ad-3B locus, and this interpretation
supports the conclusions drawn from the kinetic studies. In summary, with
genetic analysis by means of homology tests, it seems possible to determine
the extent of genetic damage in individual ad-3d mutations by the failure of
various combinations to show allelic complementation and the extent of the
functional inactivation (or spreading effect) by the growth rates of the ad-3-*
mutations in combination with reparable mutants at other loci in the immediately
adjacent regions. The genetic damage need not be precisely homologous when
nonallelic irreparable mutants fail to complement. However it is reasonable
to assume that at a minimum they each possess some genetic damage affecting
the same cistron.
Genetic analyses involving closely linked markers in the immediately adjacent
regions
Homology tests between ad-3dn mutations and reparable mutants at other
closely linked loci represent one of the most critical tests in the analysis
of ad-3IR mutents. If the apparent reduction of functional activity of loci
in the vicinity of an irreparable mutation is indeed analagous to the spreading
effects associated with variegated-type position effects in Drosophila, then
the intensity of the effect should decrease with increasing map distance and
not be present in tests with unlinked markers. Many closely linked
morphological and biochemical markers are known both to the left and to the
right of the ad-3 region (Perkins 1959, de Serres unpublished). Homology tests
with such markers require strains heterokaryon-compatible with mutants induced
in & 74A genetic background in addition to forcing biochemical and identifying
morphological markers as shown in Figure 4. The same general procedure
(F-4)
shown for LGI markers is used in complementation tests with unlinked markers.
so that such tests are unequivocal it is important that components II and III
of the trikaryon be heterozygous for at least one marker in addition to the one
being tested and homozygous for the markers al-2, cot and pan-2. Because
components II and III are always heterozygous for the inos marker, it is
possible to look for the presence of cot colonies both on supplemented and
IR
unsupplemented medium (with reference to the marker at the locus being tested).
This is to insure that this particular combination of genomes can make a cot
dikaryon, and that the absence of cot colonies cannot occur for other reasons
(reversion of the cot marker, or loss by somatic recombination, etc., in either
component II or III).
For example, to perform homology tests with a reparable mutant at the
lys-4 locus some 1.3 map units to the left (de Serres unpublished), a
trikaryon is made between the dikaryon containing an ad-3Amutation and the
appropriately marked strain containing the lys-4 marker on minimal medium.
The trikaryon 18 then plated on minimal medium supplemented with pan (calcium
pantothenate) and minimal medium supplemented with lysine and pan, and the
plates are incubated at 35-37° C. Neither component II nor component III can
form cot colonies on either of these media, but the dikaryon between them will
form cot colonies only on the doubly supplemented medium if the genetic damage
m the ad-3A-R covers the lys-4 locus, and on both media if it does not. If
the genetic damage in the ad-3Amutation is not homologous to that found in
the lys-4 mutant than the functional activity of the wild type lys-4 locus can
be determined by measuring the linear growth rate of the cot colonies at 20° C.
Appropriately marked strains for such tests with mutants at the hist-2,
lys-li, hist-3 loci to the left and the nic-2 locus to the right have been
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(F-5)
synthesized only recently and only preliminary data are currently available.
The results of homology tests based on the appearance of cot colonies from
the different trikaryons are shown in Figure 5. The genetic damage in none
of the ad-3B+" mutants analyzed was homologous to that in the nic-2 marker,
but the genetic damage in the ad-3A_* mutants 13-0 and 122-0 was homologous
to that found in the hist-3 marker. No information is available on the
linear growth rates. Although incomplete, these preliminary results show
quite clearly that the genetic damage in ad-3AR mutations can extend either
to the left and/or the right of the ad-3A locus.
IR
Correlations between complementation map position and the growth rates of
ad-3-* mutants in forced dikaryons
There is a correlation between the position and extent of individual
ad-3-mutants on the complementation map (Figure 5) and the rates of growth
of ad-3* + ad-3-" combinations in forced dikaryons (Table 1). Mutants 13-0
and 122-0 show more extensive homology with other mutants than 5-0, 6-0 and
8-0, and 13-0 and 122-0 show much slower growth rates in the forced dikaryong.
The same type of correlation existe among the ad-3Bmutants. Mutants 10-1
and 121-1 show more extensive homology than mitant 2-1 or 7-) and the former
show much slower rates of growth for heterokaryons with either reparable or
irreparable ad-3A mutants. The correlation 18 by no means complete since
123-1 is indistinguishable from 2-1 and 7-1 on the basis of present homology
tests. It is possible that any correlation between the total extent of genetic
damage and the intensity of the spreading effect at a particular linked genetic
loci is obscured because individual ad-3B mutations have not been mapped on
the right. By mapping these mutants more precisely it may be possible to
determine whether the various types of interaction observed in the forced
each of the two components.
Detection of ad-3
mutations with marked spreading effects on loci in the
The rationale of the approach used to screen large numbers of ad-3-K
mutations for those with the most extreme spreading effects 18 that such
mutations should show a marked delay in the time of the appearance of a
positive response in the heterokaryon tests used to determine genotype.
Ad-3A"* or ad-3Bmutations with extreme spreading effects will appear to
be ad-3A ad-3B double mutants at the times the tests are normally scored (2 or
3 days) and positive reactions with the ad-3A or ad-ZB tester will occur at
some later time. Heterokaryon tests were made on 34 X-ray-induced ad-3A**
and 68 X-ray-induced ad-3BR mutations to screen for such mutations, and we
found that the reaction of 2 of the ad-3BIR mutations with the ad-3AP tester
was delayed until at least 5-7 days after inoculation (Webber and de Serres,
unpublished). Honology tests between these two mutants and the ad-3A+R
mutations recovered from this same experiment (de Serres, unpublished) have
shown homology for all pairwise combinations tested. This 18 interpreted as
indicating that the genetic damage in these ad-3B+" mutants covers the entire
region between the ad-3A and ad-3B loci, so that even those ad-3A-K mutations
that have resulted from chromosome breaks in close proximity to the ad-ZA
locus will show homology. This type of correlation suggests that the spreading
effects associated with ad-34mutations show a gradient in the intensity of
their effects in Neurospora as in Drosophila. These additional data provide
even more striking evidence that the functional activity of the wild type ad-3A
locus in ad-3B+" mutants depends on proximity to the site of the chromosome
damage in the ad-3BIR mutant.
'
New approaches for determining the mechanism of the spreading effects
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associated with irreparable ad-3 mutations
The influence of the spreading effects associated with ad-34* mutations
on nonallelic complementation between them and reparable mutants at closely
linked loci may well depend on (1) the organization of the chromosome, (2)
the mechanism of information transfer from DNA to messenger RNA, (3) the
mechanism of the translation of this message on the ribosone, (4) the mechanism
by which the polypeptide chain is "activated" and transformed into "functional
protein," and (5) whether the reparable mutants do or do not show allelic
complementation. One might well ask whether the growth rate of such dikaryons
is limited by the activity of the wild type locus (in component II) of the
linked reparable mutant being tested (in component III) (see Figure 3) or
whether the growth of the dikaryon is dependent in the activity of an
interaction product. The type of interaction that can take place between the
gene products specified by homologous loci in each component of these dikaryons
will depend on whether a.llelic complementation is restricted to protein-protein
interaction (Woodward 1959, Fincham and Coddington 1.963) or whether it can
take place at other levels of organization (Zipser and Perrin 1962).
The ad-3A locus is ideally situated for more detailed analysis of the
mechanism of the spreading effects associated with certain ad-3A%* mutations.
It is close proximity on the left (about 0.3-0.5 map units) to the histo)
operon (Giles 1964) and closely linked on the right (about 0.1-0.3 map units)
to the ad-3B locus. Mutants in the hist-3 operon show allelic complementation
and the complementation map consists of at least 14 complementation units or
complons (Webber 1964a). Mutants at the ad-ZB locus also show allelic
I
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2.7
complementation and the complementation map consists of at least 17
complons (de Serres 1964b and unpublished). A wide variety of complementing
mutants with restricted complon coverage are available in both sets of
mutants, so that it will be possible to perform tests for nonallelic
complementation between various ad-3A_* mutations with complementing mutants
on different parts of the complementation map. From the work on Drosophila
there should be a gradient in the spreading effect across the ad-3B locus or
the hist-3 operon so that the strength of the response obtained with different
complementing mutants in tests for nonallelic complementation may depend on
their location on the complementation map.
Any noteworthy effect on nonallelic complementation observed with the
hist-3 testers can be correlated with changes in biochemical activity. That
part of the hist-3 operon adjacent to the ad-3 region 18 believed to be that
controlling histidinol dehydrogenase activity (Webber 19640) with the other
two biochemical activities located further to the left. A comparison of the
histidinol dehydrogenase activity (1) of dikaryons between hist-3 testers
lacking this activity in combination with ad-3A+ mutations with (2) that of
dikaryons between hist-% testers possessing this activity should provide
information for the interpretation of the mechanism of the spreading effect
phenomenon. If, however, the hist-3 operon 18 a coordinate unit of
transcription with the genetic information in the entire hist-3 region
transcribed into a single, polycistronic messenger RNA as proposed by Giles
and Ahmed (Giles 1964, Ahmed et al. 1964), one might predict that the use of
various complementing hist-3 mitants in such experiments would be without
effect. We have no data from such trails as yet since critical experiments
require the use of genetically marked strains of hist-3 mutants that are
still in preparation. This approach should provide significant information,
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nevertheless, on whether (1) weak nonallelic complementation between
ad-3ATR and hist-3 mutants can be attributed to a reduction in histidinol
dehydrogenase activity, and (2) whether normal nonallelic complementation
can be restored by providing a source of this enzyme activity in the other
component of the dikaryon.
tvu radost
* Street Atimingas
::.
LITERATURE CITED
1. Ahmed, A., M. E. Case and N. H. Giles 1964 The nature of complementation
among mutants in the histidine-3 region of Neurospora crassa. Cold
Spring Harbor Symp. Quant. Biol. 29: (in press). ·
2. Ames, B. N. and P. E. Hartman 1962 In: The molecular basis of neoplasia,
. p. 322-345. The Univ. of Texas Press, Austin.
3. Ames, B. N. and P. E. Hartman 1963 The histidine operon. Cold Spring
Harbor Symp. Quant. Biol. 28: 349-356.
4. Atwood, K. C. and F. Mukai 1953 Indispensable gene functions in Neurospora.
Proc. Natl. Acad. Sci. U. 8. 39: 1027-1035.
5. Brockman, H. E. and F. J. de Serres 1963 Induction of ad-3 mutants of
Neurospora crassa by 2-aminopurine. Genetics 48: 597-604.
6. de Serres, F. J. 1956 Studies with purple adenine mutants in Neurospora
crassa. I. Structural and functional complexity in the ad-3 region.
Genetics 41: 668-676.
7. de Serres, F. J. 1962 Heterokaryon-incompatibility factor interaction in
tests between Neurospora mutants. Science 138: 1342-1343.
8. de Serres, F. J. 1964a Genetic analysis of the structure of the ad-3
region of Neurospora crassa by means of irreparable recessive lethal
mutations. Genetics 50: 21-30.
9. de Serres, F. J. 19640 Mutagenesis and chromosome structure. J. Cellular
Comp. Physiol. Supplement (in press).
10. de Serres, F. J. and H. G. Kølmark 1959 A direct method for determination
of forward-mutation rates in Neurospora crassa. Nature 182: 1249-1250.
11. de Serres, F. J. and R. S. Osterbind 1962 Estimation of the relative
frequencies of X-ray-induced viable and recessive lethal mutations in the
ad-3 region of Neurospora crassa. Genetics 47: 793-796.
12. Fincham, J. R. S, and A. Coddington 1963 The mechanism of complementation
between an mutants of Neurospora crassa. Cold Spring Harbor Symp. Quant.
Biol. 28: 517-527.
13. Garnjobst, L. 1953 Genetic control of heterocaryosis in Neurospora crassa.
Am. J. Botany 40: 607-614.
14. Garnjobst, L. 1955 Further analysis of genetic control of heterocaryosis
in Neurospora crassa. Am. J. Botany 42: 448-448.
15. Giles, N. H. 1964 Genetic fine structure in relation to function in
Neurospora. In: Genetics Today Symposium 1: p. 17-30. Proc. llth
Inter. Congr. Genetics. S. J. Geerts, Editor, Pergamon Press, Oxford.
16. Hartman, P. E., J. D. Loper, and D. Serman 1960 Fine structure mapping
by complete transduction between histidine-requiring Salmonella mutants.
The Journal of General Microbiology 22: 323-353,
17. Holloway, B. W. 1955 Genetic control of heterocaryosis in Neurospora
crassa. Am. J. Botany 39: 574-577.
18. Jacob, F. and J. Monod 1961 Ón the regulation of gene activity. Cold
Spring Harbor Symp. Quant. Biol. 26: 193-211. .
19. Lewis, E. B. 1950 The phenomenon of position effect. Adv. Genetics 3:
73-115.
20. Lukens, L. N. and J. M. Buchanan 1959 Biosynthesis of purines. XIV.
The enzymatic synthesis of 5-amino-1-ribosyl-4-imidazolecarboxylic acid
5'-phosphate from 5-amino-2-ribosylimidazole 5'-phosphate and carbon
dioxide. J. Biol. Chem. 234: 1799-1805.
21. McClintock, B. 1956 Controlling elements and the gene. Cold Spring
Harbor Symp. Quant. Biol. 21: 197-216.
Perkins, D. D. 1959 New markers and multiple point linkage data in
Neurospora. Genetics W4: 1185-1208.
23. Pittenger, T. H. and T. G. Brawner 1961 Genetic control of nuclear
selection in Neurospora heterokaryons. Genetics 46: 1645-1663.
24. Ryan, F. J., G. W. Beadle and E. L. Tatum 1943 The tube method of
measuring growth rate of Neurospora. Am. J. Botany 30: 784-799.
25. Webber, B. B. 1959 Comparative complementation and genetic maps of the
hist-3 locus in Neurospora crassa. Genetics 44: 543-544.
26. Webber, B. B. 19648 Genetic and biochemical studies of histidine-requiring
mutants of Neurospora crassa. III. Correspondence between biochemical
characteristics and complementation map position of hist-3 mutants.
Genetics (in press).
27. Webber, B. B. 19648 Genetic and biochemical studies of histidine-requiring
mutants of Neurospora crassa. IV. Linkage relationships of hist-3
mutants. Genetics (in press).
28. Webber, B. B. and F. J. de Serres 1964 Induction kinetics and genetic
analysis of X-ray-induced mutations in the ad-3 region of Neurospora crassa.
Proc. Natl. Acad. Sci. (in press).
29. Woodward, D.O., C. W. H. Partridge and N. H. Giles 1958 Complementation
at the ad-4 locus in Neurospora crasse. Proc. Natl. Acad. Sci. V. S.
44: 1237-1244.
30. Woodward, D. 0. 1959 Enzyme complementation in vitro between
adeny losuccinaseless mutants of Neurospora crassa. Proc. Natl. Acad.
Sci. U. s. 45: 846-850.
31. Zipser, D. and D. Perrin 1963 Complementation on ribosomes. Cold Spring
Harbor Symp. Quant. Biol. 28: 533-537.
Table 1
Genetic Composition of Each Component of the Dikaryon Used
in Forward-Mutation Experiments
Genotype of Each Component
Component I
(Strain Number 74-0R60-29A) (Strain Number 74-0R31-16A)
Linkage Group
IR
ad-2
cot
inos
pan-2
Genetic markers are as follows: A - mating type; hist-2 - histidine-requiring;
ad-3A, ad-3B, ad-2 - adenine-requiring; nic-2 - niacin-requiring; al-2 -
albino mycelium and conidia; cot - temperature-sensitive morphological (slower
growth the higher the temperature); inos - inositol-requiring; pan-
pantothenate-requiring.
Tablo 2
Mean Linear Growth Rates (nom nart) of Porced Dikaryons between Strains
REPARABLE OR IRREPARABLES
Carrying that Recessive Lethal Mutations (on Minimal Medium + 2 y/mi
Calcium Pantothenate - 23°c)
ad-3B
IR
11-1-2
IR
11-1-7
IR
11-1-10
IR
11-1-121
IR R
11-1-12311-1-
11-1.5
2.26+0.03
2.100.05
1.70£0.02
1.99+0.02
1.7310.06
2.80+0.04
IR
11-1-6
2.29+0.08
2.1910.03
1.52+0.10
1.7940.08
1.00+0.20
2.83+0.05
IR
1.62£0.12
1.58+0.04
1.41+0.04
1.9410.13
2.80+0.04
11-1-8 2.1810.04
IR
11-1-23 2.24+0.06
IR
11-1-122 2.31+0.06
1.56+0.31
1.81+0.16
2.7710.04
1.42+0.07
-
0.32+0.02
1.08+0.04
2.58+0.14
(11-1-11 2.8610.05
2.80+0.07
2.72+0.05
2.59+0.18
2.55+0.26
2.9230.08
IR = Irreparable recessive lethal mutation on minimal + adenine or complete
medium,
REPARABLE RECESSIVE LETHAL
R = Bobine mutation on minimal + adenine or complete medium.
. - Combination not viable as a dikaryon.
* = Quadruplicate growth tubes for each dikaryon, 95% confidence limits given
for each estimate.
LINDS
Figure 1. Procedure for obtaining forced dikaryons between nonallelic
reparable or irreparable mutations at the ad-3A locus (ad-3A*) with reparable
or irreparable mutations at the ad-2B locus (ad-38*) (200dified from de Serres
1964a).
Figure 2. Forward-mutation frequencies for ad-3 and ad- 3INmutations
after exposure to 250 Kv X rays (modified from Webber and de Serres 1964).
Figure 3. Complementation map of X-ray-induced reparable and irreparable
recessive lethal mutations in the ad-3 region showing the extent and type of
functional damage in individual mutations: (Solid bars - irreparable recessive
lethal mutations; irregular ends indicate unknown limits; cross-hatched bars •
reparable recessive lethal mutations.) (Adapted from de Serres 1964.)
Figure 4. Procedure to determine ihe extent of genetic damage and
functional inactivation in irreparable ad-3 mutations with the use of genetic
markers at loci in the immediately adjacent genetic regions. The procedure is
illustrated for tests with a marker at the lys-4 locus (Lysine-requiring).
Figure 5. Complementation map of X-ray-induced reparable and irreparable
recessive lethal mutations in the ad-3 region showing the extent and type of
functional damage in individual ad-3A_K mutations in the regions to the left
and to the right of the ad-3A cistron. (Solid bars = irreparable recessive
lethal mutations; irregular ends indicate unknown limits; cross-hatched
bars # reparable recessive lethal mutations.) (Strain 14-1-4 is a reparable
but noncomplementing hist-3 mutant.)
Mgure 1
R.
Dikamyon 1
I 'A h1st-2 ad-3A" ad-38* nic-2 + , ad-2, +, 1n06, +
II A + ad-3A + + al-2, +,cot, +, pan-2

Dikaryon 2
I' A hist-2 ad-3An ad-3BR nic-2 +, ad-2, +, 1906, +
II' A + + ad-38% + al-2, + ,cot, +, pan-2
-
Trikaryon
I + I' A hist-2 ad-34* ad-35* nic-2 + , ad-2, + , ino8, +
II A + ad-3A + + al-2, +,cot, +, pan-2
LII' A + + ad-33% + al-2, + ,cot,' +, pan-2
- Forced Dikaryon
A + ad-3a* + + al-2, + ,cot, +, pan-2
A +, + ad-38% + al-2, + ,cot, + , pan-2

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1
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