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Differential transcription of nonrepeated DNA during development of Neurospora crassa
DEVELOPMENTAL
BIOLOGY
43, 35-41
Differential
(1975)
Transcription Development
Department
of Nonrepeated of Neurospora
S. K. DUTTA AND R. K. of Botany, Howard University, Accepted
November
DNA during
crassa
CHAUDHURI Washington
D.C.
20001
I, 1974
The transcription of non-repeated DNA sequences of four developmental cell types of the eukaryotic fungus Neurospora crassa was measured quantitatively by DNA:RNA hybridization using hydroxyapatite chromatography. DNA transcription was 34% (68% of the genome) at midlog phase of mycelial growth and 15% at conidial phase. Conidial sprouts and unbranched mycelial cells showed markedly increased transcription, 25% and 27% respectively. DNAs from mycelia and conidia did not show any difference in nucleotide base composition and sequence homologies, It was concluded that approximately 30% of N. crassa genome consist of sequences which are involved either in cell differentiation and development.
tive measures of differential gene expression in eukaryotic cell types (Hahn and Laird, 1971; Grady and Campbell, 1973; Gelderman et al. 1971). In this paper we present data on differential gene expression in these different cell types of N. crassa as studied by DNA: RNA hybridization in formamide solution and using hydroxyapatite chromatography (Britten and Kohne, 1968). Marked differences of transcription in different stages of development were observed.
INTRODUCTION
N. crassa, a primitive eukaryotic fungus, has several distinct cell types such as conidia, mycelia, ascospores and others. Conidial cells germinate and then undergo different phases of growth in liquid culture before growing into mycelia as described below: (a) Stationary phase-conidial cells represent the stationary or dormant phase, (b) Conidial sprouts-4-6 hr after germination of conidia in shake culture sprouts appear as small protrusions under the microscope; (c) Unbranched myceliasprouted conidia grow into unbranched myCelia after 8-10 hr shake culture, and finally (d) Fully grown branched mycelia-which develop 18-20 hr after conidial germination. These four differentiated cell types are distinctly dissimilar. They can be easily grown in large quantities in shake culture and allowing isolation of nucleic acids from specified stages which is essential for our studies with RNA-driven reaction solution. An illustration of growth phase a, b, c, and d is given in Fig. 1. These developmental phases of N. crassa provide an unique opportunity to study the control of transcription and differential gene expression in an eukaryote. Estimates of transcript ion of non-repeated DNA sequences have provided quantita-
MATERIALS
METHODS
N. crassa 74A wild type strain, obtained from Fungal Genetics Stock Center (FGSC #987), Humboldt, CA was used throughout this study. Four different cell types, which represent different developmental stages (as illustrated in Fig. 1) of N. crassa, were harvested as described below. Conidia were grown on Vogel’s (1956) agar medium harvested by straining through fourfold cheese cloth and washed with distilled water. Sprouted conidial cells were grown with aeration for 4-6 hr at 30°C. These sprouted cells were sieved through fourfold cheese cloth. Unbranched mycelia were grown in liquid shake culture for 8 hr at 30°C. All of these cells were harvested by centrifugation at 2000 g. 35
CopyiRht 0 197.5 hy Academlr Press. Inc. All rights of reproduction in anv form reserved.
AND
36
DEVELOPMENTAL
BIOLOGY
VOLUME
43. 1975
FI IG. 1. Representative photographs of different developmental cell types of N. crassa used by us for these studi ies. Each of these cell types were grown under specific conditions and microscopic pictures were taken with Ziess camera filter microscope. Figure IA and 1B were 720x magnified. whereas 1C and 1D were 650 x lified respectively. Several fields were viewed in every case and they were found to be uniform as she bwn in the photographs.
Branched mycelial cells were harvested 18-20 hr after inoculation by straining through fourfold cheese cloth and were well squeezed before use. of total cellular RNA. Total Isolation cellular RNA was extracted according to the method of Kohne and Byers (1973), slightly modified and adopted for N. crassa. Large mass of N. crassa mycelial cells at midlog phase of growth were ground in an specially made grinding mill (Weiss et al., 1970). The mixture of lysing solution contained 0.50 M Tris-HCl in order to maintain the pH of the homogenate between 7.2 and 7.5. Conidial cells were broken by passing through a needle valve with a pressure drop of 30,000 psi using a motor driven hydraulic press (Aminco, Bethesda, MD). Preparation of unlabeled and 3”P-labeled nonrepeated DNA. Unlabeled DNA was isolated from lyophilised mycelia and 32P-
labeled DNA was extracted from harvested mycelia according to the method described by Dutta and Ojha (1972). Preparation of nonrepeated DNA was as described by Dutta (197:3). 32P-labeling of N. crassa conidial DNA is described by Mandisodza and Dutta (1971). Laheled DNAs were passed through a needle valve with a pressure drop of 50,000 psi (50 K sheared) as stated above. between Procedures for hybridization 32P-DNA and total cellular RNA. 32Plabeled DNA and total cellular RNA were denatured together at 80°C for 6-8 min in the presence of 50% formamide and incubated at 35°C with 0.4 M phosphate buffer (PB) pH 7.0. 0.6 M NaCl, 0.01 M EDTA (adopted from the procedure used by Dr. B. H. Hoyer of Carnegie Institution, Washington, D.C.-personal communication). Less than 2 pg 32P-labeled DNA and
DUTTA
AND CHAUDHURI
Changing
considerably higher amount (about 18-20 mg/ml) of RNA were used so that reaction would occur mainly between DNA and complementary RNA and not between DNA and DNA. After incubation, the annealing solution was passed through hydroxyapatite (HA) column previously equilibrated at 60°C with 0.24 M PB pH 6.8 and 0.4?% SDS. The column was then washed with 0.14 A4 phosphate buffer to remove any sodium lauryl sulphate in the column. The material that remained adsorbed to the column represented DNA:RNA hybrids which were eluted with 0.48 M phosphate buffer. To test that true DNA:RNA hybrid formation occurred the following procedures were adopted. The hybrid duplexes were dialysed extensively against 0.01 M PB, pH 6.8 and incubated with pancreatic RNase -A (50 pg/ml) from Calbiochem and RNase T, (20 unit/ml) from Sigma Chemicals at 37°C for 1 hr. The mixture was then passed through a HA column equilibrated with 0.14 M PB pH 6.8 and 0.4% SLS at 60°C and the column was washed with the same solution to remove 32P single strand DNAs which were adsorbed by the column. This single stranded DNA eluted represented the amount of DNA which had hybridized with whole RNA. As described elsewhere (Dutta 1973) no appreciable DNA:RNA reactions obtained when these single strand 3ZP-DNAs were hybridized with whole RNA from Escherichia coli under identical conditions indicating specificity of N. crussa DNA and RNA. As described by Kohne and Byers (1973) and also as observed by us (Dutta and Chandhuri). presence of RNase does not interfere with adsorption of DNA to HA. The other test for DNA:RNA hybrid formation consisted of thermal stability studies of homo- and heteroduplexes as described by Dutta and Ojha (1972). RESULTS
Nonrepeated DNA characteristics. 50 K sheared N. crass-a denatured DNA was
Transcription
37
in Development
annealed at 2.5 Cot (moles. liter-‘. sec. same as M. L- ‘.S.) in 0.48 M phosphate buffer at 65°C. The HA chromatography of the annealing mixtures revealed the existence of about 887 slow reassociating, i.e. unique (nonrepeated) DNA. The typical second order DNA:DNA reassociation kinetics of this nonrepeated DNA suggests absence of any repetitive fraction. 32P-labeled whole DNA, which contains both repetitive and nonrepetitive fractions, does not show this second order reassociation kinetics (Dutta, 1973). Similarities of DNAs from differentiated cells. DNA:DNA reactions using 32P-labeled DNAs of both mycelia and conidia are given in Table 1. Thermal denaturation studies (Fig. 2) of DNAs from conidia and mycelia showed typical bimodal curves. Similarly, thermal elution (Fig. 3) studies of 32P-labeled DNAs of both conidTABLE I SUMMARY OF DNA:DNA REACTIOXS USING 3YP-DNAs OF BOTH CONIDIAL AND MYCELIAI. CELLS OF N. crassa
Mycelia Conidia
Mycelia Conidia Conidia Mycelia
92 90 90 89
100 98 100 99
90.00 89.50 90.00 89.00
0.5 0.5 0.2 0.5
’ 32P-labeled DNAs (180.000-200.000 cpm/pg DNA) showed negligible self reaction (less than 0.5(%). and necessary corrections were made in every experiment. All the reactions were done in 0.48 M phosphate buffer (PB) pH 6.8. at 65°C and the corresponding C,t (mol X set/L) values at 0.12 M PB were calculated. A C,t of at least 1000 of unlabeled DNA was given in each reaction for a virtually complete reassociation. The DNA:DNA hybridization values of 92F in the case of homologous DNA of mycelia and 90’7’ in the case of homologous DNA of conidia were regarded as 1007. The corresponding heterologous reactions were normalized on this basis. ‘e50 was estimated from the thermal elution profile and is described as the temperature at which 50%, of the total “2P-labeled DNA remain as hybrids with an unlabeled DNA. The A’ e50 was determined by comparing the ‘e50 from the radioactive elution with its corresponding optical density data (internal standard).
38
DEVELOPMENTAL OPTICAL ll\ELIlZlG CURVE cf L,YCELIAL AYD CONIDIAL DhA 13 N CRASSA
.J! r
0
BIOLOGY
0
Cc6 hlYCtllAL DluA CONIDIAL DhA
A
l
a@
FIG. 2. Optical melting curves of DNAs in 0.12 M phosphate buffer using Gilford 2400 spectrophotometer. Two components of DNAs of high AT (Tm-82”C) and high GC (Tm-90.5”C) regions are evident in both mycelial and conidial cells. 100 r 90 1 SO--
VOLUME
43, 1975
total RNA pool has been determined by RNA driven saturation experiments in this laboratory (Dutta, 1973). The saturation (Table 2) level was reached at RNA Cot (M. L-‘.S.) of 8.6 x 10’. Our findings indicate that excess RNA concentration above 40 mg/ml actually retard the rate of reassociation. Under our reaction conditions, the optimum reassociation rate was observed with 18-20 mg/ml of incubation mixture. Such RNA concentration gave RNA Cot values of 8.6 x lo4 at which maximum DNA:RNA hybridization obtained was 34% as shown in Table 2. At the same Cot value, but at 40 mg/ml RNA only 22% of DNA:RNA reaction was observed. The self reactions at the Cot values (0.0050.008) were negligible (less than 0.05%). Ninety six percent DNA:DNA self reactions, and no-reaction of 32P N. crassa DNA with E. co/i RNA served as adequate control reactions to show the sensitivity of the procedures. The formamide procedure for DNA:RNA reaction allowed TABLE
2
SUMMARY OF ESTIMATES OF DNA:RNA SATURATION REACTIONS OF 32P-L~~~~~~ NONREPEATED MYCEUAI. DNA WITH RNAs FROM DIFFERENT CELL TYPES” Cell type 3ZP-DNA (RNA I
FIG. 3. Thermal elution profiles of 3*P-labeled conidial and mycelial DNAs. Double stranded native DNAs were adsorbed in hydroxyapatite in 0.14 M PB at 60°C and unadsorbed single strands were washed with 0.14 M PB and 0.4% SLS at 60°C. The temperature of the column was then raised in five degree increments until it reached 100°C. The column was allowed to be warmed for 2-3 min and two 5 ml washings were made at each temperature. This elutes 32Punique-DNA as it becomes single stranded with the raise of temperatures. Tm values (where 50% DNAs dissociate) were 90°C in both DNAs.
ial and mycelial cells did not show any difference in their dissociation pattern. DNA:RNA reactions. The amount of nonrepetitive transcripts present in the
Conidia
Sprout
DNA
Mycelia
1 Mycelia
~ E. co/i
Cot
I
L
0.005 0.007 0.008 0.008 0.005 0.005 0.005 0.006 0.006, 0.005 0.005
I
RNA
/ 1 ~ / 1
/
C,i
00 1,200 15,250 55,300 86.500 90,500 80.300 86,000 90,500 80,100 86.000 86,400 90,000
t :
Percent hybridization 96 0.05 11.5 22 33 34 13 14 15 22 25 25.5 00.75
1
“All reactions were done at 35°C with 50% formamide in 0.6 M NaCl, 0.4 M PB and 0.01 A4 EDTA for varying times needed to reach the required RNA C,t values. Nonrepeated 32P-labeled DNA (300,000~ 350,000 cpmlpg DNA) was isolated as described in the text.
DUTTA
AND CHAUDHURI
Changing
us to run the reaction for a longer period at a lower temparature. Self reaction control values for unique DNA samples used in this study were obtained by incubating the DNAs with same amount of RNAs, as used in respective DNA-RNA reactions, which had been first hydrolyzed by alkali. Such addition of alkali-digested and subsequently dialysed RNAs to reaction mixtures, had no effect on the rates of reassociation of the unique DNA samples as well. This provided additional evidence that our RNA samples did not contain contaminating DNA. Hybridization of RNA from different developmental stages. Table 3 summarizes the extent of hybridization of 32P-labeled non-repeated DNA from mycelial cells at mid log phase and RNA from different developmental stages of N. crassa. Maximum transcription of N. crassa DNA was observed in mid log phase (i.e 18-20 hr) of growth. Figure 4 summarizes data of the thermal stability of heteroduplexes beTABLE SUMMARY
OF EXPRESSION
Bl
C)
Conidia Sprout Unbranched Mycelia (18 Conidia Sprout Unbranched Mycelia (18 Conidia Sprout Unbranched Mycelia (18
Whole
mycelia hr old)
8.8 8.6 8.6 8.6
mycelia hr old)
mycelia hr old)
8.6 8.6 8.6 8.6
RNA
x x x x -
10’ 10’ 10” 10’
x x x x
10’ 10’ 10’ 10’
C,t
39
in Development
tween 32P-DNA of the mid log phase myCelia1 cells and RNA from other developmental stages. There is no significant difference of the ‘e50 (i.e., when 50% of hybrid molecules remain double stranded) of DNA:RNA hybrids using RNAs of different developmental stages. These hybrids had high thermal stability indicating a negligible proportion of mismatched bases in the hybrids. DISCUSSION
We have shown in this paper (Table 3) that the maximum number (34% of single stranded nonrepeated DNAs) of N. crassa genes were transcribed during the mid log phase of mycelial growth. About 50% of these genes are not transcribed in conidial cells, which are regarded to be at stationary phase. Other cells at varying growth show intermediate levels of transcription between these two extremes. Validity of these data depends on satisfactory answers to some questions related to these 3
OF NONREPEATED 32P-DNA FROM MYCELIAL CELLS AT MIDLOC DIFFERENT DEVELOPMENTAL STAGESO
Cell type
Al
Transcription
3ZP-Unique DNA C,t
0.78 1.72 1.72 0.80 1 X 1om3 1 X 10-s 1 X 10-Z 1 X 10-Z 1.2 X 10-z 4 x 10-Z 2.4 x 10m3 1 X 1o-2
PHASE WITH RESPECT TO
Unlabeled total DNA C,t 1320 1560 1320 1560 -
SZP-DNA adsorbed in HA column (per cent) 15 25 27 34 82 88 96 91 0.5 0.1 0.2 0.7
“All reactions were done for 220 hours at 35°C with 50% formamide in 0.6M NaCl and O.OlM EDTA. Nonrepeated 32P-labeled DNA (270,006380,000 cpm/pg DNA) was isolated at C,t value of 2.5 as described in the text. Unlabeled RNA and 3zP DNA concentration in reaction mixtures were 18-20 mg/ml and 0.13-0.15 &ml respectively. A) 32P-labeled nonrepeated DNA incubated with whole RNA; Bl Unadsorhed 32P-labeled nonrepeated DNA from reaction A hybridized with excess of unlabeled whole DNA from the same mycelial source. C) Unadsorbed 32P-labeled nonrepeated DNA hybridized with whole RNA from the same mycelial source. Details are described in the text under Discussion. Data presented for parts B and C prove conclusively that each of the DNA:RNA reactions in section A were complete.
40
DEVELOPMENTAL
Lo
BIOLOGY
50
; :
40
a” E
30
z =
20
IO
” u60
65
70
75
SO Temperature
85
90
35
100
“C-
FIG. 4. Thermal stability profiles of reassociated 32P-labeled homoduplexes of non-repeated mycelial DNAs (0) and heteroduplexes of 32P-labeled nonrepeated mycelial DNA and RNAs of mycelia (0) and RNAs from different developmental stages. RNADNA hybrids were adsorbed in hydroxyapatite in 0.14 PB, pH 6.8 at 60°C. The temperature of the column was then raised in five degrees increments until it reached 100°C. Then column was allowed to be warmed for 2 min and two 5 ml washings were made at each temperature. This elutes 32P-unique DNA as it becomes single stranded with the raise of temperature.
studies. These questions are: (a) Whether the individual cell types were pure, (b) whether the nonrepeated DNA sequences used by us were free from any repeated DNA sequences, and (cl whether the estimates of DNA:RNA hybridizations obtained in these studies were true. The developmental cell types used by us were pure as shown in Fig. 1. Unlike prokaryotic cells, no large scale synchronization of growth of differentiated cell types in N. crassa has so far been possible to the best of our knowledge. The conidial cells were highly resistant to rupture under the normal homogenizing procedures for myCelia1 cells used in these experiments. We had to use 30,000 psi pressure to rupture tough conidial cells. This suggests that any contamination by conidial cellular components in the mycelial DNA or RNA preparations was practically impossible. More-
VOLUME
43. 1975
over the use of fourfold cheese cloth eliminated contamination of conidial sprouts with branched mycelial cells as was routinely verified under the microscope. Secondly the nonrepeated DNA preparation showed typical second order reassociation kinetics (Dutta, 1973). 32PDNAs recovered from several DNA:RNA hybrids never showed any appreciable DNA:DNA reactions at a DNA C,t lower than five. At this DNA C,t, isolated repeated DNA of N. crassa showed 96% or more hybridization and unfractionted whole DNA showed approximately 1%15Y hybridization. Furthermore the “‘es0 of non-repeated DNA was significantly higher (88°C) than that of repeated DNA (82°C) of N. crassa (Dutta, 1973). The answer to the third question regarding dependibility of our estimates of DNA: RNA hybridization as a measure of transcription is given by the following arguments. (1) Data presented in Tables 2 and 3 suggest that all the DNA:RNA reactions were complete. Table 2 indicates that at a wRNA CJ of 8.6 K lo4 almost all of the DNA:RNA reactions were saturated. Section A of Table 3 shows the maximum RNA:DNA reaction estimates using saturation levels of whole RNA C,t values, at which self reactions of DNAs were negligible. Integrity of unreacted DNAs from reactions in section A (Table 3) is shown by data in section B. These unreacted DNAs gave maximum possible reassociation values when reacted with excess unlabeled DNAs. This also shows that the DNAs remained unaltered in the process of multiple hybridization cycle used in these experiments. Section C shows that unreacted DNAs from reactions in section A did not show appreciable hybridizations when reacted again with whole RNAs, using the same C,t values as in section A. The unreassociated DNAs from the reactions in section A remain unaltered in repeated hybridization procedures but do not hybridize with whole RNAs when reincubated under identical reaction condi-
DUTTA
AND
CHAL-DHC.HI
Chngin
tions as in section A. Theref’ore, the DNA: RNA reactions in section A were complete and represent essentially the estimates of transcription. (2) DNA:RNA reaction USing f’ormamide and phosphate huf’f’er solutions without formamide (Dutta. 1973) were found to he similar. RNA driven saturation experiments conducted by both these methods showed essentially the same values. Similar observations have also been reported by other workers (Liarakas et al., 1973). (3) Thermal stahilitl studies of’ DNA:RNA hybrids (Fig. 4) showed a high degree of complementarity within these heteroduplexes. Our estimates of’ Ifi-X4’?. DNA transcript ion were much more contrasting when compared to only 7-9“; transcription estimated in N. crnssa by Mahadevan and Bhagwat (1974). Their estimates of DNA: RNA reactions were done using total DNA, and not nonrepeated DNA sequences used by us. A general opinion seems to he that some types of’ RNA molecules are always synthesized and some RNA molecules are synthesized only at certain phases of’ growth. If’ 68’:; of’ the N. cmssa genome were transcribed into poly-A-containing mRNA precursor molecules of’ average size .500,000 dalton (Lodish et al., 1973) then N. crassa genome would contain approximately 1~5.000-25.000genes. This is based on the fact that the N. crnssa genome is 8.2 times greater than the genome size of’ E. colt’ (Chattopadhyay et al., 1972). Therefore. approximately 5OOObXOOOgenes of’ N. crassa are involved in production of’ new RNA molecules at dif’f’erent developmental stages. An enormous complexity in gene regulation. leading to dit’f’erentiation of’ this primitive eukaryotic organism, N. crassa, is therefore, apparent. This research was supported in part by a contract No. AT(40-1) 4182with the L1.S. Atomic Energy Commission and No. 10t5-5X5 with the U.S. Department of iYava1 Research. We are grateful to Dr. Johnson ChOppaIa of Howard University. for his help in taking
g Transcription
in Development
41
picturesof differentialcell typesof N. crassa.Weare grateful
to Drs.
B. H. Hoyer
and I. B. Dawid,
Depart-
ments of Terrestrial Magnetismand Embryology, CarnegieInstitution of WashingtonD. C., and Dr. R. Ray of HowardUni\rersity.WashingtonD.C. for their valuable
suggestions
in preparation
of the manuscript.
REFERENCES BRITTEN.
sequences
R. .J., AND KOHNE. D. E. (1968). Repeated in DNA. Science 161, 529-5,57
CHATTOPADHYAY,
S.
K..
KOHSE.
D.
E..
ANI)
DI.TTA,
S. K. (197:!). Isolation and characterization ofrRXA genes in N. CTGS,XX. proc. .Vat. Acad. %i. 1T.S.A. 63 ‘3266-‘1259. , , D&A, S. K. (1973). Transcription of nonrepeated DNA in N. crassa. Riochim. Riophys. Acta 324, 482Li87. DUTTA, S. K., AND OJHA, M. (1972). Relatedness hetween major taxonomic groups of fungi hased on the measurement of DNA nucleotide sequence homology. Mol. Cm. Genetics 114, 232-240. GEI.IX?KMAN, A. N.. RAKE, A. V., and BHITTEN, R. J. (19711. Transcription of nonrepeated DNA in neonatal and fetal mice. Proc. Nat/. Acad. Sci. C.S.A. 68, 172-176. GHADY, I,. ,J., and CAMPHELI, W’. P. (1973. Nonrepetitive DNA transcription in mouse ceils grorvn in tissue culture. Nature ~‘Vleu, Hial. 243, 19n-198. HAHN, W. E.. and LAIRD, C. D. (19’ill. Transcription of nonrepeated DNA in mouse brain. Scrience 173, 1,5%162. KOHNE, D. E., and BYEKS, M. J. (19’7:3). Amplification and evolution of Deoxyribonucleic Acid Sequences Expressed as Rihonucleic Acid. Biochemi.str>, 12, 237:ih”:m. LIARAKOS, C. D., ROSE>, J. M., and (I‘MALLEY, B. W. (1973). Effect of Estrogen on Gene Expression of Chick Tritiated Unique Deoxyribonucleic Acid as measured hy Hybridization in Rihonucleic Acid. Excess. Rmhemist~~ 12, X309-2916. Lomstl, H. F., FIKTEI+ R. A.. and JACORSOS, A. (1973). Transcription and structure of the genome of the cellular slime Mold Dicot>ste/ium discoideum. Symposia on Quantitative Biology. Cold Spring Harbor, XXXVIII, 899-914. MAHADEVAS. P. R., and BHAG4WAT. A. S. (1974). Control of transcription in N. cr~ssc~. Basic tiff Sci. 3 , 22%2’39.< M~sn~so~z.~, M. T., and DUTTA, S. K. (1971). jzP. labeling of’ N. crassa conidial DNA. Neurosporn ,Veu,s/etter 18, 1”. VOGEI.. H. .J. (1956). A convenient growth medium for Neurospora. Microbioi. Genet. h/L. 13, 42LG. W’EISS, H. VON JACOW, G., KLIN(;E.~WKG, M., and BLzcftE& T. (1970). Characterization of N. crossa mitochondria prepared with a grind mill. Europ. J. Hwchem. 14, 7,%8’.
BIOLOGY
43, 35-41
Differential
(1975)
Transcription Development
Department
of Nonrepeated of Neurospora
S. K. DUTTA AND R. K. of Botany, Howard University, Accepted
November
DNA during
crassa
CHAUDHURI Washington
D.C.
20001
I, 1974
The transcription of non-repeated DNA sequences of four developmental cell types of the eukaryotic fungus Neurospora crassa was measured quantitatively by DNA:RNA hybridization using hydroxyapatite chromatography. DNA transcription was 34% (68% of the genome) at midlog phase of mycelial growth and 15% at conidial phase. Conidial sprouts and unbranched mycelial cells showed markedly increased transcription, 25% and 27% respectively. DNAs from mycelia and conidia did not show any difference in nucleotide base composition and sequence homologies, It was concluded that approximately 30% of N. crassa genome consist of sequences which are involved either in cell differentiation and development.
tive measures of differential gene expression in eukaryotic cell types (Hahn and Laird, 1971; Grady and Campbell, 1973; Gelderman et al. 1971). In this paper we present data on differential gene expression in these different cell types of N. crassa as studied by DNA: RNA hybridization in formamide solution and using hydroxyapatite chromatography (Britten and Kohne, 1968). Marked differences of transcription in different stages of development were observed.
INTRODUCTION
N. crassa, a primitive eukaryotic fungus, has several distinct cell types such as conidia, mycelia, ascospores and others. Conidial cells germinate and then undergo different phases of growth in liquid culture before growing into mycelia as described below: (a) Stationary phase-conidial cells represent the stationary or dormant phase, (b) Conidial sprouts-4-6 hr after germination of conidia in shake culture sprouts appear as small protrusions under the microscope; (c) Unbranched myceliasprouted conidia grow into unbranched myCelia after 8-10 hr shake culture, and finally (d) Fully grown branched mycelia-which develop 18-20 hr after conidial germination. These four differentiated cell types are distinctly dissimilar. They can be easily grown in large quantities in shake culture and allowing isolation of nucleic acids from specified stages which is essential for our studies with RNA-driven reaction solution. An illustration of growth phase a, b, c, and d is given in Fig. 1. These developmental phases of N. crassa provide an unique opportunity to study the control of transcription and differential gene expression in an eukaryote. Estimates of transcript ion of non-repeated DNA sequences have provided quantita-
MATERIALS
METHODS
N. crassa 74A wild type strain, obtained from Fungal Genetics Stock Center (FGSC #987), Humboldt, CA was used throughout this study. Four different cell types, which represent different developmental stages (as illustrated in Fig. 1) of N. crassa, were harvested as described below. Conidia were grown on Vogel’s (1956) agar medium harvested by straining through fourfold cheese cloth and washed with distilled water. Sprouted conidial cells were grown with aeration for 4-6 hr at 30°C. These sprouted cells were sieved through fourfold cheese cloth. Unbranched mycelia were grown in liquid shake culture for 8 hr at 30°C. All of these cells were harvested by centrifugation at 2000 g. 35
CopyiRht 0 197.5 hy Academlr Press. Inc. All rights of reproduction in anv form reserved.
AND
36
DEVELOPMENTAL
BIOLOGY
VOLUME
43. 1975
FI IG. 1. Representative photographs of different developmental cell types of N. crassa used by us for these studi ies. Each of these cell types were grown under specific conditions and microscopic pictures were taken with Ziess camera filter microscope. Figure IA and 1B were 720x magnified. whereas 1C and 1D were 650 x lified respectively. Several fields were viewed in every case and they were found to be uniform as she bwn in the photographs.
Branched mycelial cells were harvested 18-20 hr after inoculation by straining through fourfold cheese cloth and were well squeezed before use. of total cellular RNA. Total Isolation cellular RNA was extracted according to the method of Kohne and Byers (1973), slightly modified and adopted for N. crassa. Large mass of N. crassa mycelial cells at midlog phase of growth were ground in an specially made grinding mill (Weiss et al., 1970). The mixture of lysing solution contained 0.50 M Tris-HCl in order to maintain the pH of the homogenate between 7.2 and 7.5. Conidial cells were broken by passing through a needle valve with a pressure drop of 30,000 psi using a motor driven hydraulic press (Aminco, Bethesda, MD). Preparation of unlabeled and 3”P-labeled nonrepeated DNA. Unlabeled DNA was isolated from lyophilised mycelia and 32P-
labeled DNA was extracted from harvested mycelia according to the method described by Dutta and Ojha (1972). Preparation of nonrepeated DNA was as described by Dutta (197:3). 32P-labeling of N. crassa conidial DNA is described by Mandisodza and Dutta (1971). Laheled DNAs were passed through a needle valve with a pressure drop of 50,000 psi (50 K sheared) as stated above. between Procedures for hybridization 32P-DNA and total cellular RNA. 32Plabeled DNA and total cellular RNA were denatured together at 80°C for 6-8 min in the presence of 50% formamide and incubated at 35°C with 0.4 M phosphate buffer (PB) pH 7.0. 0.6 M NaCl, 0.01 M EDTA (adopted from the procedure used by Dr. B. H. Hoyer of Carnegie Institution, Washington, D.C.-personal communication). Less than 2 pg 32P-labeled DNA and
DUTTA
AND CHAUDHURI
Changing
considerably higher amount (about 18-20 mg/ml) of RNA were used so that reaction would occur mainly between DNA and complementary RNA and not between DNA and DNA. After incubation, the annealing solution was passed through hydroxyapatite (HA) column previously equilibrated at 60°C with 0.24 M PB pH 6.8 and 0.4?% SDS. The column was then washed with 0.14 A4 phosphate buffer to remove any sodium lauryl sulphate in the column. The material that remained adsorbed to the column represented DNA:RNA hybrids which were eluted with 0.48 M phosphate buffer. To test that true DNA:RNA hybrid formation occurred the following procedures were adopted. The hybrid duplexes were dialysed extensively against 0.01 M PB, pH 6.8 and incubated with pancreatic RNase -A (50 pg/ml) from Calbiochem and RNase T, (20 unit/ml) from Sigma Chemicals at 37°C for 1 hr. The mixture was then passed through a HA column equilibrated with 0.14 M PB pH 6.8 and 0.4% SLS at 60°C and the column was washed with the same solution to remove 32P single strand DNAs which were adsorbed by the column. This single stranded DNA eluted represented the amount of DNA which had hybridized with whole RNA. As described elsewhere (Dutta 1973) no appreciable DNA:RNA reactions obtained when these single strand 3ZP-DNAs were hybridized with whole RNA from Escherichia coli under identical conditions indicating specificity of N. crussa DNA and RNA. As described by Kohne and Byers (1973) and also as observed by us (Dutta and Chandhuri). presence of RNase does not interfere with adsorption of DNA to HA. The other test for DNA:RNA hybrid formation consisted of thermal stability studies of homo- and heteroduplexes as described by Dutta and Ojha (1972). RESULTS
Nonrepeated DNA characteristics. 50 K sheared N. crass-a denatured DNA was
Transcription
37
in Development
annealed at 2.5 Cot (moles. liter-‘. sec. same as M. L- ‘.S.) in 0.48 M phosphate buffer at 65°C. The HA chromatography of the annealing mixtures revealed the existence of about 887 slow reassociating, i.e. unique (nonrepeated) DNA. The typical second order DNA:DNA reassociation kinetics of this nonrepeated DNA suggests absence of any repetitive fraction. 32P-labeled whole DNA, which contains both repetitive and nonrepetitive fractions, does not show this second order reassociation kinetics (Dutta, 1973). Similarities of DNAs from differentiated cells. DNA:DNA reactions using 32P-labeled DNAs of both mycelia and conidia are given in Table 1. Thermal denaturation studies (Fig. 2) of DNAs from conidia and mycelia showed typical bimodal curves. Similarly, thermal elution (Fig. 3) studies of 32P-labeled DNAs of both conidTABLE I SUMMARY OF DNA:DNA REACTIOXS USING 3YP-DNAs OF BOTH CONIDIAL AND MYCELIAI. CELLS OF N. crassa
Mycelia Conidia
Mycelia Conidia Conidia Mycelia
92 90 90 89
100 98 100 99
90.00 89.50 90.00 89.00
0.5 0.5 0.2 0.5
’ 32P-labeled DNAs (180.000-200.000 cpm/pg DNA) showed negligible self reaction (less than 0.5(%). and necessary corrections were made in every experiment. All the reactions were done in 0.48 M phosphate buffer (PB) pH 6.8. at 65°C and the corresponding C,t (mol X set/L) values at 0.12 M PB were calculated. A C,t of at least 1000 of unlabeled DNA was given in each reaction for a virtually complete reassociation. The DNA:DNA hybridization values of 92F in the case of homologous DNA of mycelia and 90’7’ in the case of homologous DNA of conidia were regarded as 1007. The corresponding heterologous reactions were normalized on this basis. ‘e50 was estimated from the thermal elution profile and is described as the temperature at which 50%, of the total “2P-labeled DNA remain as hybrids with an unlabeled DNA. The A’ e50 was determined by comparing the ‘e50 from the radioactive elution with its corresponding optical density data (internal standard).
38
DEVELOPMENTAL OPTICAL ll\ELIlZlG CURVE cf L,YCELIAL AYD CONIDIAL DhA 13 N CRASSA
.J! r
0
BIOLOGY
0
Cc6 hlYCtllAL DluA CONIDIAL DhA
A
l
a@
FIG. 2. Optical melting curves of DNAs in 0.12 M phosphate buffer using Gilford 2400 spectrophotometer. Two components of DNAs of high AT (Tm-82”C) and high GC (Tm-90.5”C) regions are evident in both mycelial and conidial cells. 100 r 90 1 SO--
VOLUME
43, 1975
total RNA pool has been determined by RNA driven saturation experiments in this laboratory (Dutta, 1973). The saturation (Table 2) level was reached at RNA Cot (M. L-‘.S.) of 8.6 x 10’. Our findings indicate that excess RNA concentration above 40 mg/ml actually retard the rate of reassociation. Under our reaction conditions, the optimum reassociation rate was observed with 18-20 mg/ml of incubation mixture. Such RNA concentration gave RNA Cot values of 8.6 x lo4 at which maximum DNA:RNA hybridization obtained was 34% as shown in Table 2. At the same Cot value, but at 40 mg/ml RNA only 22% of DNA:RNA reaction was observed. The self reactions at the Cot values (0.0050.008) were negligible (less than 0.05%). Ninety six percent DNA:DNA self reactions, and no-reaction of 32P N. crassa DNA with E. co/i RNA served as adequate control reactions to show the sensitivity of the procedures. The formamide procedure for DNA:RNA reaction allowed TABLE
2
SUMMARY OF ESTIMATES OF DNA:RNA SATURATION REACTIONS OF 32P-L~~~~~~ NONREPEATED MYCEUAI. DNA WITH RNAs FROM DIFFERENT CELL TYPES” Cell type 3ZP-DNA (RNA I
FIG. 3. Thermal elution profiles of 3*P-labeled conidial and mycelial DNAs. Double stranded native DNAs were adsorbed in hydroxyapatite in 0.14 M PB at 60°C and unadsorbed single strands were washed with 0.14 M PB and 0.4% SLS at 60°C. The temperature of the column was then raised in five degree increments until it reached 100°C. The column was allowed to be warmed for 2-3 min and two 5 ml washings were made at each temperature. This elutes 32Punique-DNA as it becomes single stranded with the raise of temperatures. Tm values (where 50% DNAs dissociate) were 90°C in both DNAs.
ial and mycelial cells did not show any difference in their dissociation pattern. DNA:RNA reactions. The amount of nonrepetitive transcripts present in the
Conidia
Sprout
DNA
Mycelia
1 Mycelia
~ E. co/i
Cot
I
L
0.005 0.007 0.008 0.008 0.005 0.005 0.005 0.006 0.006, 0.005 0.005
I
RNA
/ 1 ~ / 1
/
C,i
00 1,200 15,250 55,300 86.500 90,500 80.300 86,000 90,500 80,100 86.000 86,400 90,000
t :
Percent hybridization 96 0.05 11.5 22 33 34 13 14 15 22 25 25.5 00.75
1
“All reactions were done at 35°C with 50% formamide in 0.6 M NaCl, 0.4 M PB and 0.01 A4 EDTA for varying times needed to reach the required RNA C,t values. Nonrepeated 32P-labeled DNA (300,000~ 350,000 cpmlpg DNA) was isolated as described in the text.
DUTTA
AND CHAUDHURI
Changing
us to run the reaction for a longer period at a lower temparature. Self reaction control values for unique DNA samples used in this study were obtained by incubating the DNAs with same amount of RNAs, as used in respective DNA-RNA reactions, which had been first hydrolyzed by alkali. Such addition of alkali-digested and subsequently dialysed RNAs to reaction mixtures, had no effect on the rates of reassociation of the unique DNA samples as well. This provided additional evidence that our RNA samples did not contain contaminating DNA. Hybridization of RNA from different developmental stages. Table 3 summarizes the extent of hybridization of 32P-labeled non-repeated DNA from mycelial cells at mid log phase and RNA from different developmental stages of N. crassa. Maximum transcription of N. crassa DNA was observed in mid log phase (i.e 18-20 hr) of growth. Figure 4 summarizes data of the thermal stability of heteroduplexes beTABLE SUMMARY
OF EXPRESSION
Bl
C)
Conidia Sprout Unbranched Mycelia (18 Conidia Sprout Unbranched Mycelia (18 Conidia Sprout Unbranched Mycelia (18
Whole
mycelia hr old)
8.8 8.6 8.6 8.6
mycelia hr old)
mycelia hr old)
8.6 8.6 8.6 8.6
RNA
x x x x -
10’ 10’ 10” 10’
x x x x
10’ 10’ 10’ 10’
C,t
39
in Development
tween 32P-DNA of the mid log phase myCelia1 cells and RNA from other developmental stages. There is no significant difference of the ‘e50 (i.e., when 50% of hybrid molecules remain double stranded) of DNA:RNA hybrids using RNAs of different developmental stages. These hybrids had high thermal stability indicating a negligible proportion of mismatched bases in the hybrids. DISCUSSION
We have shown in this paper (Table 3) that the maximum number (34% of single stranded nonrepeated DNAs) of N. crassa genes were transcribed during the mid log phase of mycelial growth. About 50% of these genes are not transcribed in conidial cells, which are regarded to be at stationary phase. Other cells at varying growth show intermediate levels of transcription between these two extremes. Validity of these data depends on satisfactory answers to some questions related to these 3
OF NONREPEATED 32P-DNA FROM MYCELIAL CELLS AT MIDLOC DIFFERENT DEVELOPMENTAL STAGESO
Cell type
Al
Transcription
3ZP-Unique DNA C,t
0.78 1.72 1.72 0.80 1 X 1om3 1 X 10-s 1 X 10-Z 1 X 10-Z 1.2 X 10-z 4 x 10-Z 2.4 x 10m3 1 X 1o-2
PHASE WITH RESPECT TO
Unlabeled total DNA C,t 1320 1560 1320 1560 -
SZP-DNA adsorbed in HA column (per cent) 15 25 27 34 82 88 96 91 0.5 0.1 0.2 0.7
“All reactions were done for 220 hours at 35°C with 50% formamide in 0.6M NaCl and O.OlM EDTA. Nonrepeated 32P-labeled DNA (270,006380,000 cpm/pg DNA) was isolated at C,t value of 2.5 as described in the text. Unlabeled RNA and 3zP DNA concentration in reaction mixtures were 18-20 mg/ml and 0.13-0.15 &ml respectively. A) 32P-labeled nonrepeated DNA incubated with whole RNA; Bl Unadsorhed 32P-labeled nonrepeated DNA from reaction A hybridized with excess of unlabeled whole DNA from the same mycelial source. C) Unadsorbed 32P-labeled nonrepeated DNA hybridized with whole RNA from the same mycelial source. Details are described in the text under Discussion. Data presented for parts B and C prove conclusively that each of the DNA:RNA reactions in section A were complete.
40
DEVELOPMENTAL
Lo
BIOLOGY
50
; :
40
a” E
30
z =
20
IO
” u60
65
70
75
SO Temperature
85
90
35
100
“C-
FIG. 4. Thermal stability profiles of reassociated 32P-labeled homoduplexes of non-repeated mycelial DNAs (0) and heteroduplexes of 32P-labeled nonrepeated mycelial DNA and RNAs of mycelia (0) and RNAs from different developmental stages. RNADNA hybrids were adsorbed in hydroxyapatite in 0.14 PB, pH 6.8 at 60°C. The temperature of the column was then raised in five degrees increments until it reached 100°C. Then column was allowed to be warmed for 2 min and two 5 ml washings were made at each temperature. This elutes 32P-unique DNA as it becomes single stranded with the raise of temperature.
studies. These questions are: (a) Whether the individual cell types were pure, (b) whether the nonrepeated DNA sequences used by us were free from any repeated DNA sequences, and (cl whether the estimates of DNA:RNA hybridizations obtained in these studies were true. The developmental cell types used by us were pure as shown in Fig. 1. Unlike prokaryotic cells, no large scale synchronization of growth of differentiated cell types in N. crassa has so far been possible to the best of our knowledge. The conidial cells were highly resistant to rupture under the normal homogenizing procedures for myCelia1 cells used in these experiments. We had to use 30,000 psi pressure to rupture tough conidial cells. This suggests that any contamination by conidial cellular components in the mycelial DNA or RNA preparations was practically impossible. More-
VOLUME
43. 1975
over the use of fourfold cheese cloth eliminated contamination of conidial sprouts with branched mycelial cells as was routinely verified under the microscope. Secondly the nonrepeated DNA preparation showed typical second order reassociation kinetics (Dutta, 1973). 32PDNAs recovered from several DNA:RNA hybrids never showed any appreciable DNA:DNA reactions at a DNA C,t lower than five. At this DNA C,t, isolated repeated DNA of N. crassa showed 96% or more hybridization and unfractionted whole DNA showed approximately 1%15Y hybridization. Furthermore the “‘es0 of non-repeated DNA was significantly higher (88°C) than that of repeated DNA (82°C) of N. crassa (Dutta, 1973). The answer to the third question regarding dependibility of our estimates of DNA: RNA hybridization as a measure of transcription is given by the following arguments. (1) Data presented in Tables 2 and 3 suggest that all the DNA:RNA reactions were complete. Table 2 indicates that at a wRNA CJ of 8.6 K lo4 almost all of the DNA:RNA reactions were saturated. Section A of Table 3 shows the maximum RNA:DNA reaction estimates using saturation levels of whole RNA C,t values, at which self reactions of DNAs were negligible. Integrity of unreacted DNAs from reactions in section A (Table 3) is shown by data in section B. These unreacted DNAs gave maximum possible reassociation values when reacted with excess unlabeled DNAs. This also shows that the DNAs remained unaltered in the process of multiple hybridization cycle used in these experiments. Section C shows that unreacted DNAs from reactions in section A did not show appreciable hybridizations when reacted again with whole RNAs, using the same C,t values as in section A. The unreassociated DNAs from the reactions in section A remain unaltered in repeated hybridization procedures but do not hybridize with whole RNAs when reincubated under identical reaction condi-
DUTTA
AND
CHAL-DHC.HI
Chngin
tions as in section A. Theref’ore, the DNA: RNA reactions in section A were complete and represent essentially the estimates of transcription. (2) DNA:RNA reaction USing f’ormamide and phosphate huf’f’er solutions without formamide (Dutta. 1973) were found to he similar. RNA driven saturation experiments conducted by both these methods showed essentially the same values. Similar observations have also been reported by other workers (Liarakas et al., 1973). (3) Thermal stahilitl studies of’ DNA:RNA hybrids (Fig. 4) showed a high degree of complementarity within these heteroduplexes. Our estimates of’ Ifi-X4’?. DNA transcript ion were much more contrasting when compared to only 7-9“; transcription estimated in N. crnssa by Mahadevan and Bhagwat (1974). Their estimates of DNA: RNA reactions were done using total DNA, and not nonrepeated DNA sequences used by us. A general opinion seems to he that some types of’ RNA molecules are always synthesized and some RNA molecules are synthesized only at certain phases of’ growth. If’ 68’:; of’ the N. cmssa genome were transcribed into poly-A-containing mRNA precursor molecules of’ average size .500,000 dalton (Lodish et al., 1973) then N. crassa genome would contain approximately 1~5.000-25.000genes. This is based on the fact that the N. crnssa genome is 8.2 times greater than the genome size of’ E. colt’ (Chattopadhyay et al., 1972). Therefore. approximately 5OOObXOOOgenes of’ N. crassa are involved in production of’ new RNA molecules at dif’f’erent developmental stages. An enormous complexity in gene regulation. leading to dit’f’erentiation of’ this primitive eukaryotic organism, N. crassa, is therefore, apparent. This research was supported in part by a contract No. AT(40-1) 4182with the L1.S. Atomic Energy Commission and No. 10t5-5X5 with the U.S. Department of iYava1 Research. We are grateful to Dr. Johnson ChOppaIa of Howard University. for his help in taking
g Transcription
in Development
41
picturesof differentialcell typesof N. crassa.Weare grateful
to Drs.
B. H. Hoyer
and I. B. Dawid,
Depart-
ments of Terrestrial Magnetismand Embryology, CarnegieInstitution of WashingtonD. C., and Dr. R. Ray of HowardUni\rersity.WashingtonD.C. for their valuable
suggestions
in preparation
of the manuscript.
REFERENCES BRITTEN.
sequences
R. .J., AND KOHNE. D. E. (1968). Repeated in DNA. Science 161, 529-5,57
CHATTOPADHYAY,
S.
K..
KOHSE.
D.
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ANI)
DI.TTA,
S. K. (197:!). Isolation and characterization ofrRXA genes in N. CTGS,XX. proc. .Vat. Acad. %i. 1T.S.A. 63 ‘3266-‘1259. , , D&A, S. K. (1973). Transcription of nonrepeated DNA in N. crassa. Riochim. Riophys. Acta 324, 482Li87. DUTTA, S. K., AND OJHA, M. (1972). Relatedness hetween major taxonomic groups of fungi hased on the measurement of DNA nucleotide sequence homology. Mol. Cm. Genetics 114, 232-240. GEI.IX?KMAN, A. N.. RAKE, A. V., and BHITTEN, R. J. (19711. Transcription of nonrepeated DNA in neonatal and fetal mice. Proc. Nat/. Acad. Sci. C.S.A. 68, 172-176. GHADY, I,. ,J., and CAMPHELI, W’. P. (1973. Nonrepetitive DNA transcription in mouse ceils grorvn in tissue culture. Nature ~‘Vleu, Hial. 243, 19n-198. HAHN, W. E.. and LAIRD, C. D. (19’ill. Transcription of nonrepeated DNA in mouse brain. Scrience 173, 1,5%162. KOHNE, D. E., and BYEKS, M. J. (19’7:3). Amplification and evolution of Deoxyribonucleic Acid Sequences Expressed as Rihonucleic Acid. Biochemi.str>, 12, 237:ih”:m. LIARAKOS, C. D., ROSE>, J. M., and (I‘MALLEY, B. W. (1973). Effect of Estrogen on Gene Expression of Chick Tritiated Unique Deoxyribonucleic Acid as measured hy Hybridization in Rihonucleic Acid. Excess. Rmhemist~~ 12, X309-2916. Lomstl, H. F., FIKTEI+ R. A.. and JACORSOS, A. (1973). Transcription and structure of the genome of the cellular slime Mold Dicot>ste/ium discoideum. Symposia on Quantitative Biology. Cold Spring Harbor, XXXVIII, 899-914. MAHADEVAS. P. R., and BHAG4WAT. A. S. (1974). Control of transcription in N. cr~ssc~. Basic tiff Sci. 3 , 22%2’39.< M~sn~so~z.~, M. T., and DUTTA, S. K. (1971). jzP. labeling of’ N. crassa conidial DNA. Neurosporn ,Veu,s/etter 18, 1”. VOGEI.. H. .J. (1956). A convenient growth medium for Neurospora. Microbioi. Genet. h/L. 13, 42LG. W’EISS, H. VON JACOW, G., KLIN(;E.~WKG, M., and BLzcftE& T. (1970). Characterization of N. crossa mitochondria prepared with a grind mill. Europ. J. Hwchem. 14, 7,%8’.