- Email: [email protected]
Induction of hydroxamate-sensitive respiration in Neurospora mitochondria Transcription of nuclear DNA is required
Volume gS, number 1
FEBS LETTERS
January 1978
!?NDUCTION OF HYDROXAMATE-SENSITIVE RESPIRATION MITOCHONDRIA Transcription
IN NEUROSPORA
o f n u c l e a r D N A is r e q u i r e d
David L. E D W A R D S and Barbara W. LINGER Department o f Biochemistry, Scripps Clinic and Researel, Foundation, La Jolga, CA 92037, USA
Receiv-_d 14 October 1977
1. I n t r o d u c t i o n A topic o f current interest in studies o f mito-. chondrial biogenesis is the regulatory interactions that o c c u r b e t w e e n the m i t o c h o n d r i a l arLd nuclear g e n o m e s during this process. B o t h g e n o m e s have been s h o w n t o c o n t r i b u t e p r o d u c t s t o the assembly o f the m i t o c h o n d r i a l inner m e m b r a n e [ ! ] . Previous studies have s h o w n t h a t inhibition o f m i t o c h o n d r i a l transcription. with e d f i d i u m b r o m i d e or translation with c h l o r a m p h e n i c o l leads to an increase in the specific activities o f n u m e r o u s e n z y m e s t h a t function in the m i t o c b o n d r i o n b u t are c o d e d for in the nucleus [2--5] . These e n z y m e s include : D N A - d e p e n d e n t R N A p o l y m e r a s e , elongation factors, m e t h i o n y l - t R N A transfc rmylase, r i b o s o m a l proteins and m i t o c h o n d f i a l leucyl- and p h e n y l a l a n y l - t R N & synthetases. These studies have led to *.he posttflation t h a t the m i t o c h o n dr/al go.home ac~s to regulate the nuclear g e n o m e t h r o u g a a repressor p r o t e i n that affects transcription o f the nuclear genes coding for m i t o c h o n d r i a l c o m ponents [!,2]. We ihave b e e n studying the h y d r o x a m a t e - s e n s i t i v e respiratory p a t h w a y in m i t o c h o n d r i a f r o m N e u r o s p o r a erassa. Th/s path~vay has been s h o w n to be present in respira to~-
s y s t e m w i t h one p o r t i o n being the standard c y t o c h r o m e chain and the o t h e r being the h y d r o x a r a a t e sensitive p a t h w a y o f u n k n o w n c o m p o s i t i o n [9]. We have previously s h o w n t h a t h y d r o x a m a t e sensitive respiration can be induced in wild-type cells b y inhibition o f m i t o c h o n d r i M transcription o r m i t o c h o n d f i a l translation [ 1 0 , 1 1 ] . Protein synthesis on cycloheximide-sensitive r i b o s o m e s is also required for the p r o d u c t i o n o f h y d r o x a m a t e - s e n s i t i v e respiration in such i n d u c t i o n e x p e r i m e n t s [ 8 ] . In this r e p o r t we present a s t u d y with the drug a c t i n o m y c i n - D . This drug is an inhibitor o f D N A d e p e n d e n t R N A synthesis in m a n y s y s t e m s [12] and has b e e n s h o w n to specifically inl-dbit nuclear transcription i~ zVeuros p o r a [ 1 3 , 1 4 ] . The data show t h a t transcription o f nuclear D N A is required for the p r o d u c t i o n o f hydro×amate-sensitive respiration.
2. Materials and m e t h o d s Strain i n L 8 9 6 0 1 was o b t a i n e d f r o m the Fungal Genetics S t o c k Center, H u m b o l d t State University F o u n d a t i o n , Arcata, CA 9 5 5 2 1 . Conidia f r o m the ceils were inoculated at a c o n c e n t r a t i o n o f 5 X l 0 s conidia/ml into Voge|s" m e d i u m N [7] s u p p l e m e n t e d with sucrose (2%) and inositol ( 5 0 b~glml) and grown at 30°C in a g y r o t o r y shaker for 13.5 h at 200 roy/ rain. The m y c e l i u m was collected b y filtration o n a Buchner funnel and resuspended in 20 m l 0.1 M soadum p h o s p h a t e p H 6.2 contairdng 0.5% ethy'!enediaminetetraacetic acid ( E D T A ) . i n c u b a t i o n wa:s El~evier/Arorth-t~oiland Biomedical Presx
V o l u m e 85, n u m b e r 1
FEBS LETTERS
J a n u a w 1978
carried out in this solution for 5 min at 30°(2 with
shaking. The perrneabilized cells were then collected, washed with distilled water and resuspended in Vogels' medium supplemented as above and also containing chloramphenicol (2 mg/ml) and varying amounts o f acti_n0mycin-D. Incubation was carried out at 30°C with shaking. Respiration
measurements
3o
Z o
?
o.
were made
at various times as described [113].
3. Results and discussion
| t~J
Figure 1 shows the results obtained when EDTApermeabilized cells of strain inl-89601 are treated with chloramphenicol in the presence or absence o f actinomycin-D. In the absence o f acfinomycin-D, hydroxamate-sensitive respiration is induced and can
~_~
x o c~
>-.
medium
and completely
c~a~IL----~J~ i
2
3,
~------'~
~
4
G
5
7
Fig.1. i n d u c t i o n o f hydroxama~e-sensitive r e s p k a t i o n in
Neurospora. Cells were g r o w n i n Vogels' m e d i u m N a n d permeabitLzed w i t h E D T A as described in Materials a n d m e t h o d s . T h e permeabflized cells were t h e r e s u s p e n d e d in fresh m e d i u m c o n t a i n i n g c h l o r a m p h e n i c o l (2 m g / m l ) a n d varying a m o u n t s o f a c t i n o m y c i n - D . T i m e 0 o n the figure is t h e t i m e w h e n t h e permeabiHzed ceils were r e s u s p e n d e d in fresh m e d i u m . O p e n circles: c h l o r a m p h e n i c o l o n l y n o a c f i n o m y c i n - D . Half-fflled circles: c h l o r a m p h e n i c o l pius 2.5 ~g/ml a c t i n o m y c i n - D . Filled circles: c h l o r a m p b e n i c o i plus 5.0 ~tg]ml acfinomycha-D.
by 5 gg/rrd.
This result indicates that transcription ~f nuclear DNA is required for the production of hydro×-.anatesensitive respiration in Neurospora. In additional experiments, we have attempted to demonstrate the accumulation of ttle messenger RNA for the hydroxamate-sensW,ve pathway by sequential incubation o f the cells in cycloheximide and then actinomycin-D. These experiments have not been successful and resulted in the death o f the cells. The procedure involved permeabflization and blockade with chloramphenieol and eycloheximide fb!lowed by a second permeabflizafion
0 0
be observed in the culture after approx. 3 h incubat i o n . This lag is considerably longer than that observed
in unpermeabflized cells (approx. 30 mix. [ 10] ) and is likely due to the drastic nature o f the EDTA treatment. Once hydro×araate-sensitive respiration begins to appear in the culture, however, the activity increases in a linear manner for the next several hours. T h e d a t a i n fig. 1 s h o w t h a t w h e n t h e permeabilized cells are treated with chtoramphenicol and increasing amounts o f actinomycin-D, there is marked inhibition o f hydroxamate-sensRive respkation. The activity is inhibited by 90% by 2.5/ag actinomycha-D]ml culture
io
and blockade
with chloramphenicol
pathway [ 10,11 ]. It has also been shown directly by the isolation of mutants which cannot produce the pathway even whe:n challenged with cbAoramphenicol [15]. These mutations segregate in a Mendelian manner
in crosses. Preliminary experiments
have
assorted them into two complementation groups [ 15]. The experiments with drugs described [ 10, i 1] have shown that a mitochondrial gene product regulates the production o f the hydroxamate-sensitive pathway. This product acts in a negative manner to regulate the nuclear genes involved in the production o f t h e p a t h w a y [ 11 ] . We. h a v e a l s o r e c e n t l y - d e s c r i b e d an extrachromosomal m u t a n t , cni-3, that h a s t h e
and actinomycin-D. The nuclear nature o f the structural genes for the hydroxaraate-sensifive pathway has been interred from experiments with chloramphenicol and ethidium
hydroxamate-sensifive pathway present at ati times during the growth cycle a l t h o u ~ it is not utilized in vivo [9,16]. This mutation appears to be located in
bromide
the segment of mitochondrial
which inhibit mitochondrial
translation
transcription and result in the production o f the
and
DNA that codes for
the regulatory element o f the pathway. 41
Volume 85, number 1
FEBS LETTERS
The data p,-e=-ented here strongly suggest that the r e g u l a t i o n i n t h i s s y s t e m is e f f e c t e d b y c o n t r o l l i n g t h e
transcription o f the nuclear genes which code for components o f the pathway. Our results lend more experimental support to the reports [ 1 --5,10,I 1] concet:aing such a regulatory mechanism. Although we think it unlikely, we cannot, however, role out the possibility that the mitochoridfial product regulates tee production o f proteins (proteases, nucleases) that modify some pre-existing components which are then utilized to produce hydroxamate-sensitive respiration_ T h e r e g u l a t i o n o f g e n e a c t i v i t y b y negative c o n t r o l has been well documented in bacterial systems [ 17]. In all systems that have been well studied to date, the regulation has been carried out by a protein. Previous studies from our laboratory have indicated that the mitochondrial regulatory molecule for the hydroxamak:-sensitive system acts outside of the rnitochondrion [ 11 ], The present study indicates that the r e g u l a t i o n is at t h e l e v e l o f n u c l e a r t r a n s c r i p t i o n . ~,Ve t h i n k it m o s t l i k e l y *-hat t h e r e g u l a t o r y e l e m e n t is a
proteir_ o f mitochondrial origin that acts by inhibiting the transcription o f nucIear DNA.
Acknow|edgements
This work was supported by research grant GM-24991 from the National Institute o f General Medical Sciences to D . L E . D_ L. E. is also the recipient o f a Research C--areer Development Award from the same institute.
42
.Tarmary 1978
References ~1] Schatz, G. and Mason, T. ( t 9 7 4 ) Ann. Rev. Biochem. 43, 51--87. [2l Barath, Z. and Kuntzel, H. (1972) Nature New Biol. 240, 1 9 5 - 1 9 7 . [3] Barath, Z. and Kuntzel, H. (1972) Proc. Natl. Acad. Sci. USA 69, 1371--1374. [4] Beaucharnp, P. and Gross, S. (1976) Mature 26!, 338--340. [5] Beaucharnp, P., Horn, E. and Gross, S. (1977) Proc. Nail. Acad. Sci. USA 74, 1 1 7 2 - I 1 7 6 . [6] Lambowitz, A. and Siayman, C. W. (1971) $. Bacteriol. 108, 1 0 8 7 - 1 0 9 6 . [7] Edwaxds, D. L., Kwiecinski, F. and Horstmann, J. (1973) J. Bacteriol. 114, 164--168. [8] Schonbaum, G. R., Bonnet, W. D., Jr, Storey, B. T. and Bahr, J. T. (1971) P h n t Physiol. 47, 1 2 4 - 1 2 8 . [9] Lambowitz, A., Staymau, C. W., S~ayman, C. L. and Bonner, W. D.,jr (1972) J. BioL Chem. 247, 1536--i545. [ I0] Edwards, D. L., Rosenberg, E. and Ma:oney, F. (1974) J. BioL Chem. 249, 3551--3556. [ 1 ! ] Edwaxds, D. L. and Rosenberg, E. (I 9"r6) Eur. J. Biochem. 62, 217--221. [ 12] Reich, E. and Goldbcrg, L H. (~964) it t: Progress in Nucleic Acid Research and MolecuLar B~o!ogy (Davidson, ft. N. and Colin, W. E. eds) VoL 3, pp. ~ 8 3 - 2 3 4 , Academic Press, New York. [13] Tnnler, ~. R., Terry, K. and Matchett, W. H. (i97{)) J. Bacteriol. 103,370--374. [ 14] .Gubramani~n, K. N. and Sorger, G. J. (1972) J. Bacteriol. 110, 547--553. [15] Edwards, D. L., Guzik, H. and SVaxden, J. T (1976) in: Genetics and Biogenesis o f Chloroplasts and Mitochondfia (Bucher, Th., Ne,upert, W., Sebald, W. and Werner, S. eds) pp. 865--872, ElsevierfNorth-Hol~and, Amsterdm-n. [ 16 ] Edw~xds, D. L. ( 1977) ~ : Function o f Alternative OxMascs (Dcgn, H. and Lloyd, D. eds) Pergamon Press, London in press. [17] Gots, J. and Benson, C. (1974) Ann. Rev. Genet. 8, 79--101.
FEBS LETTERS
January 1978
!?NDUCTION OF HYDROXAMATE-SENSITIVE RESPIRATION MITOCHONDRIA Transcription
IN NEUROSPORA
o f n u c l e a r D N A is r e q u i r e d
David L. E D W A R D S and Barbara W. LINGER Department o f Biochemistry, Scripps Clinic and Researel, Foundation, La Jolga, CA 92037, USA
Receiv-_d 14 October 1977
1. I n t r o d u c t i o n A topic o f current interest in studies o f mito-. chondrial biogenesis is the regulatory interactions that o c c u r b e t w e e n the m i t o c h o n d r i a l arLd nuclear g e n o m e s during this process. B o t h g e n o m e s have been s h o w n t o c o n t r i b u t e p r o d u c t s t o the assembly o f the m i t o c h o n d r i a l inner m e m b r a n e [ ! ] . Previous studies have s h o w n t h a t inhibition o f m i t o c h o n d r i a l transcription. with e d f i d i u m b r o m i d e or translation with c h l o r a m p h e n i c o l leads to an increase in the specific activities o f n u m e r o u s e n z y m e s t h a t function in the m i t o c b o n d r i o n b u t are c o d e d for in the nucleus [2--5] . These e n z y m e s include : D N A - d e p e n d e n t R N A p o l y m e r a s e , elongation factors, m e t h i o n y l - t R N A transfc rmylase, r i b o s o m a l proteins and m i t o c h o n d f i a l leucyl- and p h e n y l a l a n y l - t R N & synthetases. These studies have led to *.he posttflation t h a t the m i t o c h o n dr/al go.home ac~s to regulate the nuclear g e n o m e t h r o u g a a repressor p r o t e i n that affects transcription o f the nuclear genes coding for m i t o c h o n d r i a l c o m ponents [!,2]. We ihave b e e n studying the h y d r o x a m a t e - s e n s i t i v e respiratory p a t h w a y in m i t o c h o n d r i a f r o m N e u r o s p o r a erassa. Th/s path~vay has been s h o w n to be present in respira to~-
s y s t e m w i t h one p o r t i o n being the standard c y t o c h r o m e chain and the o t h e r being the h y d r o x a r a a t e sensitive p a t h w a y o f u n k n o w n c o m p o s i t i o n [9]. We have previously s h o w n t h a t h y d r o x a m a t e sensitive respiration can be induced in wild-type cells b y inhibition o f m i t o c h o n d r i M transcription o r m i t o c h o n d f i a l translation [ 1 0 , 1 1 ] . Protein synthesis on cycloheximide-sensitive r i b o s o m e s is also required for the p r o d u c t i o n o f h y d r o x a m a t e - s e n s i t i v e respiration in such i n d u c t i o n e x p e r i m e n t s [ 8 ] . In this r e p o r t we present a s t u d y with the drug a c t i n o m y c i n - D . This drug is an inhibitor o f D N A d e p e n d e n t R N A synthesis in m a n y s y s t e m s [12] and has b e e n s h o w n to specifically inl-dbit nuclear transcription i~ zVeuros p o r a [ 1 3 , 1 4 ] . The data show t h a t transcription o f nuclear D N A is required for the p r o d u c t i o n o f hydro×amate-sensitive respiration.
2. Materials and m e t h o d s Strain i n L 8 9 6 0 1 was o b t a i n e d f r o m the Fungal Genetics S t o c k Center, H u m b o l d t State University F o u n d a t i o n , Arcata, CA 9 5 5 2 1 . Conidia f r o m the ceils were inoculated at a c o n c e n t r a t i o n o f 5 X l 0 s conidia/ml into Voge|s" m e d i u m N [7] s u p p l e m e n t e d with sucrose (2%) and inositol ( 5 0 b~glml) and grown at 30°C in a g y r o t o r y shaker for 13.5 h at 200 roy/ rain. The m y c e l i u m was collected b y filtration o n a Buchner funnel and resuspended in 20 m l 0.1 M soadum p h o s p h a t e p H 6.2 contairdng 0.5% ethy'!enediaminetetraacetic acid ( E D T A ) . i n c u b a t i o n wa:s El~evier/Arorth-t~oiland Biomedical Presx
V o l u m e 85, n u m b e r 1
FEBS LETTERS
J a n u a w 1978
carried out in this solution for 5 min at 30°(2 with
shaking. The perrneabilized cells were then collected, washed with distilled water and resuspended in Vogels' medium supplemented as above and also containing chloramphenicol (2 mg/ml) and varying amounts o f acti_n0mycin-D. Incubation was carried out at 30°C with shaking. Respiration
measurements
3o
Z o
?
o.
were made
at various times as described [113].
3. Results and discussion
| t~J
Figure 1 shows the results obtained when EDTApermeabilized cells of strain inl-89601 are treated with chloramphenicol in the presence or absence o f actinomycin-D. In the absence o f acfinomycin-D, hydroxamate-sensitive respiration is induced and can
~_~
x o c~
>-.
medium
and completely
c~a~IL----~J~ i
2
3,
~------'~
~
4
G
5
7
Fig.1. i n d u c t i o n o f hydroxama~e-sensitive r e s p k a t i o n in
Neurospora. Cells were g r o w n i n Vogels' m e d i u m N a n d permeabitLzed w i t h E D T A as described in Materials a n d m e t h o d s . T h e permeabflized cells were t h e r e s u s p e n d e d in fresh m e d i u m c o n t a i n i n g c h l o r a m p h e n i c o l (2 m g / m l ) a n d varying a m o u n t s o f a c t i n o m y c i n - D . T i m e 0 o n the figure is t h e t i m e w h e n t h e permeabiHzed ceils were r e s u s p e n d e d in fresh m e d i u m . O p e n circles: c h l o r a m p h e n i c o l o n l y n o a c f i n o m y c i n - D . Half-fflled circles: c h l o r a m p h e n i c o l pius 2.5 ~g/ml a c t i n o m y c i n - D . Filled circles: c h l o r a m p b e n i c o i plus 5.0 ~tg]ml acfinomycha-D.
by 5 gg/rrd.
This result indicates that transcription ~f nuclear DNA is required for the production of hydro×-.anatesensitive respiration in Neurospora. In additional experiments, we have attempted to demonstrate the accumulation of ttle messenger RNA for the hydroxamate-sensW,ve pathway by sequential incubation o f the cells in cycloheximide and then actinomycin-D. These experiments have not been successful and resulted in the death o f the cells. The procedure involved permeabflization and blockade with chloramphenieol and eycloheximide fb!lowed by a second permeabflizafion
0 0
be observed in the culture after approx. 3 h incubat i o n . This lag is considerably longer than that observed
in unpermeabflized cells (approx. 30 mix. [ 10] ) and is likely due to the drastic nature o f the EDTA treatment. Once hydro×araate-sensitive respiration begins to appear in the culture, however, the activity increases in a linear manner for the next several hours. T h e d a t a i n fig. 1 s h o w t h a t w h e n t h e permeabilized cells are treated with chtoramphenicol and increasing amounts o f actinomycin-D, there is marked inhibition o f hydroxamate-sensRive respkation. The activity is inhibited by 90% by 2.5/ag actinomycha-D]ml culture
io
and blockade
with chloramphenicol
pathway [ 10,11 ]. It has also been shown directly by the isolation of mutants which cannot produce the pathway even whe:n challenged with cbAoramphenicol [15]. These mutations segregate in a Mendelian manner
in crosses. Preliminary experiments
have
assorted them into two complementation groups [ 15]. The experiments with drugs described [ 10, i 1] have shown that a mitochondrial gene product regulates the production o f the hydroxamate-sensitive pathway. This product acts in a negative manner to regulate the nuclear genes involved in the production o f t h e p a t h w a y [ 11 ] . We. h a v e a l s o r e c e n t l y - d e s c r i b e d an extrachromosomal m u t a n t , cni-3, that h a s t h e
and actinomycin-D. The nuclear nature o f the structural genes for the hydroxaraate-sensifive pathway has been interred from experiments with chloramphenicol and ethidium
hydroxamate-sensifive pathway present at ati times during the growth cycle a l t h o u ~ it is not utilized in vivo [9,16]. This mutation appears to be located in
bromide
the segment of mitochondrial
which inhibit mitochondrial
translation
transcription and result in the production o f the
and
DNA that codes for
the regulatory element o f the pathway. 41
Volume 85, number 1
FEBS LETTERS
The data p,-e=-ented here strongly suggest that the r e g u l a t i o n i n t h i s s y s t e m is e f f e c t e d b y c o n t r o l l i n g t h e
transcription o f the nuclear genes which code for components o f the pathway. Our results lend more experimental support to the reports [ 1 --5,10,I 1] concet:aing such a regulatory mechanism. Although we think it unlikely, we cannot, however, role out the possibility that the mitochoridfial product regulates tee production o f proteins (proteases, nucleases) that modify some pre-existing components which are then utilized to produce hydroxamate-sensitive respiration_ T h e r e g u l a t i o n o f g e n e a c t i v i t y b y negative c o n t r o l has been well documented in bacterial systems [ 17]. In all systems that have been well studied to date, the regulation has been carried out by a protein. Previous studies from our laboratory have indicated that the mitochondrial regulatory molecule for the hydroxamak:-sensitive system acts outside of the rnitochondrion [ 11 ], The present study indicates that the r e g u l a t i o n is at t h e l e v e l o f n u c l e a r t r a n s c r i p t i o n . ~,Ve t h i n k it m o s t l i k e l y *-hat t h e r e g u l a t o r y e l e m e n t is a
proteir_ o f mitochondrial origin that acts by inhibiting the transcription o f nucIear DNA.
Acknow|edgements
This work was supported by research grant GM-24991 from the National Institute o f General Medical Sciences to D . L E . D_ L. E. is also the recipient o f a Research C--areer Development Award from the same institute.
42
.Tarmary 1978
References ~1] Schatz, G. and Mason, T. ( t 9 7 4 ) Ann. Rev. Biochem. 43, 51--87. [2l Barath, Z. and Kuntzel, H. (1972) Nature New Biol. 240, 1 9 5 - 1 9 7 . [3] Barath, Z. and Kuntzel, H. (1972) Proc. Natl. Acad. Sci. USA 69, 1371--1374. [4] Beaucharnp, P. and Gross, S. (1976) Mature 26!, 338--340. [5] Beaucharnp, P., Horn, E. and Gross, S. (1977) Proc. Nail. Acad. Sci. USA 74, 1 1 7 2 - I 1 7 6 . [6] Lambowitz, A. and Siayman, C. W. (1971) $. Bacteriol. 108, 1 0 8 7 - 1 0 9 6 . [7] Edwaxds, D. L., Kwiecinski, F. and Horstmann, J. (1973) J. Bacteriol. 114, 164--168. [8] Schonbaum, G. R., Bonnet, W. D., Jr, Storey, B. T. and Bahr, J. T. (1971) P h n t Physiol. 47, 1 2 4 - 1 2 8 . [9] Lambowitz, A., Staymau, C. W., S~ayman, C. L. and Bonner, W. D.,jr (1972) J. BioL Chem. 247, 1536--i545. [ I0] Edwards, D. L., Rosenberg, E. and Ma:oney, F. (1974) J. BioL Chem. 249, 3551--3556. [ 1 ! ] Edwaxds, D. L. and Rosenberg, E. (I 9"r6) Eur. J. Biochem. 62, 217--221. [ 12] Reich, E. and Goldbcrg, L H. (~964) it t: Progress in Nucleic Acid Research and MolecuLar B~o!ogy (Davidson, ft. N. and Colin, W. E. eds) VoL 3, pp. ~ 8 3 - 2 3 4 , Academic Press, New York. [13] Tnnler, ~. R., Terry, K. and Matchett, W. H. (i97{)) J. Bacteriol. 103,370--374. [ 14] .Gubramani~n, K. N. and Sorger, G. J. (1972) J. Bacteriol. 110, 547--553. [15] Edwards, D. L., Guzik, H. and SVaxden, J. T (1976) in: Genetics and Biogenesis o f Chloroplasts and Mitochondfia (Bucher, Th., Ne,upert, W., Sebald, W. and Werner, S. eds) pp. 865--872, ElsevierfNorth-Hol~and, Amsterdm-n. [ 16 ] Edw~xds, D. L. ( 1977) ~ : Function o f Alternative OxMascs (Dcgn, H. and Lloyd, D. eds) Pergamon Press, London in press. [17] Gots, J. and Benson, C. (1974) Ann. Rev. Genet. 8, 79--101.