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Gene regulation in the polyploid amphibian Odontophrynus americanus
PRELIMINARY
NOTES
Argentina and Uruguay. Yet diploid populations of this species were reported only in a few localities. Most of the other populations W. BECAK and M. T. PUEYO, Serviqu de GenCtica, of 0. ameri~an~~ have been shown to be Institute ~~tanta~, Mo Pa&, Bras2 tetraploid [3, 41. Their karyotypes with 44 sugary chromosomes show 11 groups of 4 homoAmong the anurans of the family Ceratophrydidae logues each, and, in meiotic prophases, quadtwo polyploid specieshave lxxx found: Odont~p~rynus americanus (4n =44) and Ceratophrys dorsata rivalents were observed. Comparative cyto(8~= 104). Comparative m~suremen~ in the diploid photometric measurements of the DNA con0. cultripes and the tetraploid 0. americanus indicated a reduced activity or a partial inactivation of tent of Feulgen-stained preparations of sevthe geneswhich control the enzymelactate dehydro- era1 tissues confirmed the expected ratio of genaseand the hemoglobin in the tetraploid. I:2 for the diploid and tetraploid species [jl. Gene duplication seems to be a frequent feature in the evolution of multicellular organisms. Increase in DNA content along Table 1. Locrate dehydroge~~e me~~rements phylogenetic lines has been reported in sev- in cardiac wrWcte era1cases [2, 9, 111. Qabntophrynus Oduntophrynus Polyploidy is a pa~icular case of gene cu/trees(2n=22) Americas (4n = 44) duplication which produces a drastic increase Mass Mass of DNA content. It is advantageous in the heart ULDH/ heart ULDH/ No. Sex (mg) mg sense that it furnishes the species with a No. Sex (m&r>mg large amount of genetic raw material that 42,6 79.7 I Q 36.2 30.9 1 ? 125.7 68.4 30.8 59.2 may be exploited by mutation and natural selection. This confers on the organism a ii ; 50.5 45.9 f ; 53.7 45.5 higher genetical pol~mo~hism and in gen: g 148.9 62.3 56.4 56.5 4 Q 40.8 39s 40.5 47.8 Gene regulation in the polyploid amphibian ~do~t~~~~~~~ a~~~~~~~
eral, as a consequence, a wider ecologicai and geographical range. However, duplication of a set of chromosomeswould imply a doubling of the amount of products to be synthesized by the genetic material, unless a special gene control were established. The present paper reports such a type of control in a polyploid anuran speties. The Odontophrynus, belonging to genus the family Ceratoph~didae, includes several diploid specieswith 22 chromosomes: 0. cultripes, 0. occidentalis, 0. curvalhoi and 0. americanus [5, 61. 0. americanus has a wide geographical distribution Exprl Cell Res 63
including
Brazil,
f i
; ::
10 11
P 9 12 Q 13 ? 14 ?
15 ?
16
e
:z
g
17 0 18 Q
104.9 69.5 80.6 70.3 82.7 93.5 65.9 64.6 81.0
54.5
2
78.1 71.8 53.4 68.1 53.8
77.9 80.0 55.5 62.9
22.7
45.1
6 d 6
41.0 27.0
37.0 36.7
s6
53.5 17.3 47.8 27.8 41.0 46.8 38.3 62.6 ULDH = 40.1
$
11
6 6 s
13
14 15
63.2
72.7
16 17 18
48.2 67.1
94.8 63.1
19
51.0 80.3 73.0 63.5
20 21
41.4 57.9
z:
43.7 46.3 65.6 62.2 22 23 45.5 109.8 24 29.4 95.2 25 ULDH = 69.5
z
45.9 34.8 24.6 31.1 39.2 23.6 52.4 25.9
1:
12
27.6 46.3 24.4 41.3 48.7 36.0 30.6 37.2 41.2
73.9
21 6 24 d 25 6
$
s7 $
6
8 c;
6
$ d
~3
30.8
30.7 39.0 49.1 46.9 28.0 34.2 33.0 31.0
Gene regulation in ~olyploi~
Table 2. 0. &tripes (2n = 22) blood measurements No. Sex RBC
: 6s 0.881 0.976 0.277 0.345 : ; 0.657 27 cf; 0.730 0.755 z 10 11
$ 3 d
13 14 15 16 17 18 19
d 3 Q Q Q Q
12
8
Q 20 Q 21 0
0.250 0.528 0.759 0.593 0.735 0.705 0.663 0.946 0.608 0.782 0.524 0.692 0.630 0.561 MCV
MCV Ht 318 28 358 35 253 7 290 10 351 23 356 26 291 22 320 8 340 18 395 30 320 19 367 27 397 28 287 19 297 28 280 17 422 33 362 19 375 26 412 26 356 20 = 340 pm8
T
Hb
57 47 84 80 64 58 65 89 73 58 68 59 61 67
8.5 11.5 2.6 3.4 6.8
:; :; 59 63 12
MCH
1;: 94 99 104 113 86 104 91 8:3 109 5.9 99 8.0 108 105 t’: 92 8:4 89 92 i:: 103 95 115 ii*: 7:o 111 90 5.0 MCH :=101 pg
s: 42.:
RBC. red cell count in millions/mm? MCV. mean corpuscular volume in pmS; Ht,’ volume of packed red celis in ml/100 ml (hematocrit); T, hemoglobin transmittan~; Hb, hemoglobin in g 1100 ml of blood; MCH, mean corpuscular hemoglobin in pg.
Starch gel electrophoresis of an albuminlike plasma protein of the diploid 0. cultripes and the tetraploid 0. americanus was made. No allelic polymorphism at this locus was found in the diploid species,but five distinct electrophoretic phenotypes were identified among the tetraploid population [8]. In order to make an analysis of gene products in the diploids and tetraploids the enzyme lactate dehydrogenase (LDH) and the hemoglobin were studied comparatively in the species 0. cu~tr~pes (2n=22} and 0. americanus (48 = 44). Materials and methods Twenty-five specimens of 0. cultripes from Belo Horizonte (Brazil) and 25 of 0. arnericanusfrom SLO Paul0 (Brazil) were used for the lactate dehydrogenase study.
449
The specimens were dissected. the heart isolated. weighed; homogenized and centrifuged at 10 000 g for 30 min at l-2°C. Sam&s of 0.1 ml of the suoernatant were used for LDH measurements at 25”~-in a Zeiss PMQII spectrophotometer. Total LDH was measured by the reaction of lactate (90% lactate acid, Merckj to pyruvate in presence of NAD (Sigma) as a coenzyme according to the method of Amador [I]. Saturation by the use of pyruvate as substrate results in partia1 inhibition of LDH, at 20°C [12]. Twenty-one specimens of 0. cultripes from Be10 Horizonte and 37 of 0. americanusfrom Sflo Paul0 were used for erythrocyte and hemo~obin determinations. Blood was cohected by cardiac puncture, heparin beina added as anticoaaulant. The red cell count (RBE) was determined in a Coulter counter model F. Each samule of 25 ul of blood was hemolvsed in 10 ml of 50 % solution’ of NH&OH and the trarrsmittance (T) measured in a Spectronic 20 spectrophotometer at 545 nm. Concentrations of hemoglobin were obtained bv comoarison with a standard of cyanomethemo~~b~n (18 g HbjlOO ml) used in the Hospital das Clinicas of the University of Sio Paulo. The mean corpuscular hemoglobin (MCH) was determined using the formula [(Hb) g. 10-6/RBC] pg and the mean corpuscular volume (MCV) by the formula [Ht %(lO)/(RBC/mmS)] rums.
Results
Lactate dehydrogenase: The results summarized in table 1 show that in 0. cultripes there are 69.5 ULDH/mg of cardiac muscle (S.E.M. = 14.88). In 0. americanus there are 40.1 ULDH/mg of cardiac muscle (S.E. = 10.33). The ratio of LDH for both speciesis LDH am/LDH cultr = 0.58. Chemical DNA determinations in kidney tissue of the diploid 0. amer~canus and the tetraploid 0. americanus showed a DNA ratio of 1:2 per unit of weight [lo]. Assuming that a similar situation is true for cardiac muscle of 0. cultripes (2n) and 0. americu~us (4n), the LDH values obtained indicate partial inactivation or lower activity of the LDH genesin the tetraploid specieswhen compared with the diploid. Hemoglobin: The results are summarized in tables 2, 3. The mean corpuscular hemoglobin (MCH) values of 0. umericanus (4n) and 0. cultripes (2n) are 112 pg (SE; = 14.7) Exptl Cell Res 63
450
W. Begak & hf. T. Pueyo
Sex
RBC
MCV
Ht
T
:
$
0.775 0.851
387 341
29 30
55 56
9.1 8.8
117 103
parative measurements of both species confirmed, therefore, the indication of a reduced activity of the genetic material of the tetraploid 0. americanus as compared to the diploid 0. cu~tripes.
:
;
0.755 0.745
413 418
31
56 58
8.3 8.8
109 118
Discussion
i
$
0.683 0.665
466 380
26 31
60 58
;-ii
114 124
0.413 0.558
316 394
21 22
71 68
5.2 519
126 105
0.530 0.394
377 355
20 14
70 78
5.4 3.8
102 96
If gene action in the diploid and tetraploid were to and the species amount proportional of proteins synthesized ploidy would DNA, occur in a ratio of 1: 2, respectively; however, this is not the case at least for hemoglobin and cardiac LDH. Our measurements indicate a reduced activity or partial inactivation of genes in the tetraploid. However, this would not mean permanent inactivation of the same genomes in every cell of each specimen, for interaction of the four homologous geneshas been demon&rated in the electrophoretic pheno~pes for the albumin-like plasma protein [8]. Several hypothesesmight explain the results so far obtained. Random inactivation of homologue genesin each tetraploid specimen or asynchrony of genomes would result in a condition, where only a part of the genomes is active at a time. Alternatively an increase in number of the regulator genes may result in a modification of the ratio of repressors and derepressors in the tetraploid as compared to the diploid species. Yet, the same result could be obtained by a relatively smailer rate of transcription of the polyploid species.
Table 3. 0. americanus (4n =44) blood measurements No.
ii ; 1:
:
11 6 12 8 13 c3 :i $ 16 8
Ii&
MCH
0.893
459
41
46
11.0
132
0.848 0.436 0.454 0.525
400 435 462 380
34 19 21 20
54 73 69 71
9.4 4.8 :.:
110 110 123 99
0.842
451
38
51
10:2
120
17
6
0.935
406
38
50
10.5
112
18 19
6 6 8 8 5 8 8 6
0.471 0.903
488 374
23 30
49 67
10.8 6.1
129 134
20 22 21 23 24 2.5
0.813
381
31
55
9.1
111
0.832 0.626
432 479
36 30
55 57
9.1 8.5
109 135
0.660
333
22
70
5.4
82
d’
0.800 0.420 0.850
450 357 470
36 15 40
54 16 5.5
9.4 4.2 ;*:
117 100 107
27 0 28 ? 29 ?
0.515 0.451 0.345
427 266 318
22 12 11
66 76 85
4:2 2.4
122 93 1::
26
0Q
0.472 0.780
423 318
33 15
74 50
10.5 4.6
114
32 0
0.536
410
22
70
5.4
97
33 34 35
Q Q P
0.485 0.591 0.660
350 406 469
17 24 31
74 68 61
4.6 5.9 7.5
95 100 114
37 36
Q Q
0.562 0.596
402 391
22 24
62 63
7.3 7.0
129 117
31 30
MCV= 405prnS
MCH=ll2pg
and 101 pg (S.E. =4X4), respectively; thus the proportion is of MCH am/MCH cultr = 1.11. The mean corpuscular volume (MCV) for the former is 405 ,um3 (S.E.=92.5) and This work was SuDDOrted by the Fund&o de Ampare a Pesquisa do‘ Estado-de S. Paula; the Fundo for the latter 340 prns (S.E. =44.4) thus giv- de Pesauisas do InstituteButantan,theConselhoNaing the proportion of MCV am/MCV cultr = cional -de Pesquisas, and the Rockefeller Founda1.19. Linear correlation between the MCH tion. and MCV is significant for both species. REFERENCES The regression equations for 0. ~Itr~pes is MCV= IO (MCHj-680, and for 0. ameri1. Amador, E, Dorfman, L E & Wacker, W E C, Clin them 9 (1964) 391. canus is MCV = 5(MCH)-155. 2. A&ii, N B, Mattinson, G, Bc@c, W & Ohno, The hemoglobin values obtained by comS, Chromosoma 17 (196.5) 1. Exptl Cell Res 63
Presence of cellulase in the algae Scenedesmus 451
outer (pectic), middle and inner (cellulose) layers. None of the components of the layers was chemically identified. The authors observed that cell walls accumulated in the medium (CWM) as a result of autospore release from mother cells and were deprived of the inner cellulose layer. From this arose the question whether the CWM of other species of Scenedesmus, for example SC.obliquus, are also deprived of the cellulose layer. It seemedsuitable to compare the cell walls accumulated in the medium (CWM), with the native cell walls isolated Received May 20, 1970 by use of homogeni~tions (CWH); these latter possessedall the components. In an earlier work [7], the removal of the inner Presenceof cehlase in the algae Scenedesmus layer from the CWH using cellulase and heliJ. BURCZYK,’ H. GRZYBEK,P J. BANA?? and case was successful. In this case, if the ultraE. BANAS,” ‘Institute of Zootechnics at Grodziec SL, structure of CWH digested by cellulase did Laboratory of Applied Biochemistry, Gumna/Cieszyn; ~Silesian Medical School, Department of Histology and not differ from that of CWM it could beasEmbryology, Laboratory of Electronmicroscopy, Zabrze~Boki~~ca; and a~~~fitute of Zootec~~.cs,Labora- sumed to be due to the presenceof endogenic tory of Electronmicros~opy at Zator, Poland cellulolytic enzymes in SC. 0bliquu.s. Investigations of the cell wall (CW) of the The algae Scenedesmusobliauus var. altemas Christjuk unicellular fresh-water algae Scenedesmus and strain-633 used for isolation of CWH and CWM were bacte~olo~ica~v and algolo&ally clean. The C~~ore~~a(C~~orococc~Ze~) were performed to algae were fro& the collecti& Gf the Zootechnic determine their structure and to elaborate Institute in Zator. The culture conditions and methods of isolation of native CWH have been described possible mild methods of degradation. earlier [7]. CWM were obtained from 2 month cultures Until the present time the following CW by centrifuging at 800 g for 15 min. The white upper layer from the sediment was harvested with a spatula. degradation methods have been described: The thus-obtained CWM was cleaned by repeated (1) mechanical (a) homogenisation with glass centrifugation in a conical test-tube, diameter 1.0 cm, in conditions as described above. The CWM, after beads [l]; (b) high-pressure extrusion [2]; washing three times with water, was prepared observation by fixation in (2) ultrasonic [3, 4]; (3) biological (phages) for ei~~o~icros~opic KMnO, and OsO,, drying in aqueous solutions with r51. increasing concentration of methanol, embedding in It seemstheoretically possible to elaborate methacrylate and additionally contrasting with uranyl acetate. The method is described in detail in a previous a degradation method on the basis of knowl- work [7]. The thin sections were observed with an edge of the intact CW structure, by the use electron microscope JEM 7. of enzymes from egzogenic (lyse), or endogenie (autolyse) sources, as is done with a The results are presented in figs l-4 and textfig. I. From fig. 1 it can be concluded that number of bacteria. The only information concerning the CW structure of the algae Scenedesmus is given by Bisalputra et al. [6]. On the basis of histochemical and electronmicroscopical observations, the authors have distinguished three layers in the CW: the 3. Bwak, M L, BeGak, W & Rabello, M N, Chromosoma 19 (1966) 188. 4. Ibid 22 (1967)~192. 5. wak, M L, Denaro, L & Becak, W, Cytogenetics 9 (1970) 225. 6. Becak. M L. Becak. W & Vizotto, L D, Experie&la 26 (1970) 545.’ 7. Bwak, W, Bqak, M L, Lavalle, D & Schreiber, G, Chromosoma 23 (1967) 14. 8. Beqak, W, Schwantes, A R & Schwantes, M L B, J exutl zoo1 168 (1968) 473. 9. Bqak, W, Bqak, M L; Schreiber, G, Lavalle, D & Amorim. F 0. Exuerientia 26 (1970) 204. 10. Bqak, W & Goissis, G, Experientia. In press. 11. Ohno, S, Sex chromosomes and sex-linked genes. Springer, Berlin (1967). 12. Plagemann, P G W, Gregory, K F & WrobIewski, F, Biochem Z 334 (1961) 37.
Exptl CeN Res 63
NOTES
Argentina and Uruguay. Yet diploid populations of this species were reported only in a few localities. Most of the other populations W. BECAK and M. T. PUEYO, Serviqu de GenCtica, of 0. ameri~an~~ have been shown to be Institute ~~tanta~, Mo Pa&, Bras2 tetraploid [3, 41. Their karyotypes with 44 sugary chromosomes show 11 groups of 4 homoAmong the anurans of the family Ceratophrydidae logues each, and, in meiotic prophases, quadtwo polyploid specieshave lxxx found: Odont~p~rynus americanus (4n =44) and Ceratophrys dorsata rivalents were observed. Comparative cyto(8~= 104). Comparative m~suremen~ in the diploid photometric measurements of the DNA con0. cultripes and the tetraploid 0. americanus indicated a reduced activity or a partial inactivation of tent of Feulgen-stained preparations of sevthe geneswhich control the enzymelactate dehydro- era1 tissues confirmed the expected ratio of genaseand the hemoglobin in the tetraploid. I:2 for the diploid and tetraploid species [jl. Gene duplication seems to be a frequent feature in the evolution of multicellular organisms. Increase in DNA content along Table 1. Locrate dehydroge~~e me~~rements phylogenetic lines has been reported in sev- in cardiac wrWcte era1cases [2, 9, 111. Qabntophrynus Oduntophrynus Polyploidy is a pa~icular case of gene cu/trees(2n=22) Americas (4n = 44) duplication which produces a drastic increase Mass Mass of DNA content. It is advantageous in the heart ULDH/ heart ULDH/ No. Sex (mg) mg sense that it furnishes the species with a No. Sex (m&r>mg large amount of genetic raw material that 42,6 79.7 I Q 36.2 30.9 1 ? 125.7 68.4 30.8 59.2 may be exploited by mutation and natural selection. This confers on the organism a ii ; 50.5 45.9 f ; 53.7 45.5 higher genetical pol~mo~hism and in gen: g 148.9 62.3 56.4 56.5 4 Q 40.8 39s 40.5 47.8 Gene regulation in the polyploid amphibian ~do~t~~~~~~~ a~~~~~~~
eral, as a consequence, a wider ecologicai and geographical range. However, duplication of a set of chromosomeswould imply a doubling of the amount of products to be synthesized by the genetic material, unless a special gene control were established. The present paper reports such a type of control in a polyploid anuran speties. The Odontophrynus, belonging to genus the family Ceratoph~didae, includes several diploid specieswith 22 chromosomes: 0. cultripes, 0. occidentalis, 0. curvalhoi and 0. americanus [5, 61. 0. americanus has a wide geographical distribution Exprl Cell Res 63
including
Brazil,
f i
; ::
10 11
P 9 12 Q 13 ? 14 ?
15 ?
16
e
:z
g
17 0 18 Q
104.9 69.5 80.6 70.3 82.7 93.5 65.9 64.6 81.0
54.5
2
78.1 71.8 53.4 68.1 53.8
77.9 80.0 55.5 62.9
22.7
45.1
6 d 6
41.0 27.0
37.0 36.7
s6
53.5 17.3 47.8 27.8 41.0 46.8 38.3 62.6 ULDH = 40.1
$
11
6 6 s
13
14 15
63.2
72.7
16 17 18
48.2 67.1
94.8 63.1
19
51.0 80.3 73.0 63.5
20 21
41.4 57.9
z:
43.7 46.3 65.6 62.2 22 23 45.5 109.8 24 29.4 95.2 25 ULDH = 69.5
z
45.9 34.8 24.6 31.1 39.2 23.6 52.4 25.9
1:
12
27.6 46.3 24.4 41.3 48.7 36.0 30.6 37.2 41.2
73.9
21 6 24 d 25 6
$
s7 $
6
8 c;
6
$ d
~3
30.8
30.7 39.0 49.1 46.9 28.0 34.2 33.0 31.0
Gene regulation in ~olyploi~
Table 2. 0. &tripes (2n = 22) blood measurements No. Sex RBC
: 6s 0.881 0.976 0.277 0.345 : ; 0.657 27 cf; 0.730 0.755 z 10 11
$ 3 d
13 14 15 16 17 18 19
d 3 Q Q Q Q
12
8
Q 20 Q 21 0
0.250 0.528 0.759 0.593 0.735 0.705 0.663 0.946 0.608 0.782 0.524 0.692 0.630 0.561 MCV
MCV Ht 318 28 358 35 253 7 290 10 351 23 356 26 291 22 320 8 340 18 395 30 320 19 367 27 397 28 287 19 297 28 280 17 422 33 362 19 375 26 412 26 356 20 = 340 pm8
T
Hb
57 47 84 80 64 58 65 89 73 58 68 59 61 67
8.5 11.5 2.6 3.4 6.8
:; :; 59 63 12
MCH
1;: 94 99 104 113 86 104 91 8:3 109 5.9 99 8.0 108 105 t’: 92 8:4 89 92 i:: 103 95 115 ii*: 7:o 111 90 5.0 MCH :=101 pg
s: 42.:
RBC. red cell count in millions/mm? MCV. mean corpuscular volume in pmS; Ht,’ volume of packed red celis in ml/100 ml (hematocrit); T, hemoglobin transmittan~; Hb, hemoglobin in g 1100 ml of blood; MCH, mean corpuscular hemoglobin in pg.
Starch gel electrophoresis of an albuminlike plasma protein of the diploid 0. cultripes and the tetraploid 0. americanus was made. No allelic polymorphism at this locus was found in the diploid species,but five distinct electrophoretic phenotypes were identified among the tetraploid population [8]. In order to make an analysis of gene products in the diploids and tetraploids the enzyme lactate dehydrogenase (LDH) and the hemoglobin were studied comparatively in the species 0. cu~tr~pes (2n=22} and 0. americanus (48 = 44). Materials and methods Twenty-five specimens of 0. cultripes from Belo Horizonte (Brazil) and 25 of 0. arnericanusfrom SLO Paul0 (Brazil) were used for the lactate dehydrogenase study.
449
The specimens were dissected. the heart isolated. weighed; homogenized and centrifuged at 10 000 g for 30 min at l-2°C. Sam&s of 0.1 ml of the suoernatant were used for LDH measurements at 25”~-in a Zeiss PMQII spectrophotometer. Total LDH was measured by the reaction of lactate (90% lactate acid, Merckj to pyruvate in presence of NAD (Sigma) as a coenzyme according to the method of Amador [I]. Saturation by the use of pyruvate as substrate results in partia1 inhibition of LDH, at 20°C [12]. Twenty-one specimens of 0. cultripes from Be10 Horizonte and 37 of 0. americanusfrom Sflo Paul0 were used for erythrocyte and hemo~obin determinations. Blood was cohected by cardiac puncture, heparin beina added as anticoaaulant. The red cell count (RBE) was determined in a Coulter counter model F. Each samule of 25 ul of blood was hemolvsed in 10 ml of 50 % solution’ of NH&OH and the trarrsmittance (T) measured in a Spectronic 20 spectrophotometer at 545 nm. Concentrations of hemoglobin were obtained bv comoarison with a standard of cyanomethemo~~b~n (18 g HbjlOO ml) used in the Hospital das Clinicas of the University of Sio Paulo. The mean corpuscular hemoglobin (MCH) was determined using the formula [(Hb) g. 10-6/RBC] pg and the mean corpuscular volume (MCV) by the formula [Ht %(lO)/(RBC/mmS)] rums.
Results
Lactate dehydrogenase: The results summarized in table 1 show that in 0. cultripes there are 69.5 ULDH/mg of cardiac muscle (S.E.M. = 14.88). In 0. americanus there are 40.1 ULDH/mg of cardiac muscle (S.E. = 10.33). The ratio of LDH for both speciesis LDH am/LDH cultr = 0.58. Chemical DNA determinations in kidney tissue of the diploid 0. amer~canus and the tetraploid 0. americanus showed a DNA ratio of 1:2 per unit of weight [lo]. Assuming that a similar situation is true for cardiac muscle of 0. cultripes (2n) and 0. americu~us (4n), the LDH values obtained indicate partial inactivation or lower activity of the LDH genesin the tetraploid specieswhen compared with the diploid. Hemoglobin: The results are summarized in tables 2, 3. The mean corpuscular hemoglobin (MCH) values of 0. umericanus (4n) and 0. cultripes (2n) are 112 pg (SE; = 14.7) Exptl Cell Res 63
450
W. Begak & hf. T. Pueyo
Sex
RBC
MCV
Ht
T
:
$
0.775 0.851
387 341
29 30
55 56
9.1 8.8
117 103
parative measurements of both species confirmed, therefore, the indication of a reduced activity of the genetic material of the tetraploid 0. americanus as compared to the diploid 0. cu~tripes.
:
;
0.755 0.745
413 418
31
56 58
8.3 8.8
109 118
Discussion
i
$
0.683 0.665
466 380
26 31
60 58
;-ii
114 124
0.413 0.558
316 394
21 22
71 68
5.2 519
126 105
0.530 0.394
377 355
20 14
70 78
5.4 3.8
102 96
If gene action in the diploid and tetraploid were to and the species amount proportional of proteins synthesized ploidy would DNA, occur in a ratio of 1: 2, respectively; however, this is not the case at least for hemoglobin and cardiac LDH. Our measurements indicate a reduced activity or partial inactivation of genes in the tetraploid. However, this would not mean permanent inactivation of the same genomes in every cell of each specimen, for interaction of the four homologous geneshas been demon&rated in the electrophoretic pheno~pes for the albumin-like plasma protein [8]. Several hypothesesmight explain the results so far obtained. Random inactivation of homologue genesin each tetraploid specimen or asynchrony of genomes would result in a condition, where only a part of the genomes is active at a time. Alternatively an increase in number of the regulator genes may result in a modification of the ratio of repressors and derepressors in the tetraploid as compared to the diploid species. Yet, the same result could be obtained by a relatively smailer rate of transcription of the polyploid species.
Table 3. 0. americanus (4n =44) blood measurements No.
ii ; 1:
:
11 6 12 8 13 c3 :i $ 16 8
Ii&
MCH
0.893
459
41
46
11.0
132
0.848 0.436 0.454 0.525
400 435 462 380
34 19 21 20
54 73 69 71
9.4 4.8 :.:
110 110 123 99
0.842
451
38
51
10:2
120
17
6
0.935
406
38
50
10.5
112
18 19
6 6 8 8 5 8 8 6
0.471 0.903
488 374
23 30
49 67
10.8 6.1
129 134
20 22 21 23 24 2.5
0.813
381
31
55
9.1
111
0.832 0.626
432 479
36 30
55 57
9.1 8.5
109 135
0.660
333
22
70
5.4
82
d’
0.800 0.420 0.850
450 357 470
36 15 40
54 16 5.5
9.4 4.2 ;*:
117 100 107
27 0 28 ? 29 ?
0.515 0.451 0.345
427 266 318
22 12 11
66 76 85
4:2 2.4
122 93 1::
26
0Q
0.472 0.780
423 318
33 15
74 50
10.5 4.6
114
32 0
0.536
410
22
70
5.4
97
33 34 35
Q Q P
0.485 0.591 0.660
350 406 469
17 24 31
74 68 61
4.6 5.9 7.5
95 100 114
37 36
Q Q
0.562 0.596
402 391
22 24
62 63
7.3 7.0
129 117
31 30
MCV= 405prnS
MCH=ll2pg
and 101 pg (S.E. =4X4), respectively; thus the proportion is of MCH am/MCH cultr = 1.11. The mean corpuscular volume (MCV) for the former is 405 ,um3 (S.E.=92.5) and This work was SuDDOrted by the Fund&o de Ampare a Pesquisa do‘ Estado-de S. Paula; the Fundo for the latter 340 prns (S.E. =44.4) thus giv- de Pesauisas do InstituteButantan,theConselhoNaing the proportion of MCV am/MCV cultr = cional -de Pesquisas, and the Rockefeller Founda1.19. Linear correlation between the MCH tion. and MCV is significant for both species. REFERENCES The regression equations for 0. ~Itr~pes is MCV= IO (MCHj-680, and for 0. ameri1. Amador, E, Dorfman, L E & Wacker, W E C, Clin them 9 (1964) 391. canus is MCV = 5(MCH)-155. 2. A&ii, N B, Mattinson, G, Bc@c, W & Ohno, The hemoglobin values obtained by comS, Chromosoma 17 (196.5) 1. Exptl Cell Res 63
Presence of cellulase in the algae Scenedesmus 451
outer (pectic), middle and inner (cellulose) layers. None of the components of the layers was chemically identified. The authors observed that cell walls accumulated in the medium (CWM) as a result of autospore release from mother cells and were deprived of the inner cellulose layer. From this arose the question whether the CWM of other species of Scenedesmus, for example SC.obliquus, are also deprived of the cellulose layer. It seemedsuitable to compare the cell walls accumulated in the medium (CWM), with the native cell walls isolated Received May 20, 1970 by use of homogeni~tions (CWH); these latter possessedall the components. In an earlier work [7], the removal of the inner Presenceof cehlase in the algae Scenedesmus layer from the CWH using cellulase and heliJ. BURCZYK,’ H. GRZYBEK,P J. BANA?? and case was successful. In this case, if the ultraE. BANAS,” ‘Institute of Zootechnics at Grodziec SL, structure of CWH digested by cellulase did Laboratory of Applied Biochemistry, Gumna/Cieszyn; ~Silesian Medical School, Department of Histology and not differ from that of CWM it could beasEmbryology, Laboratory of Electronmicroscopy, Zabrze~Boki~~ca; and a~~~fitute of Zootec~~.cs,Labora- sumed to be due to the presenceof endogenic tory of Electronmicros~opy at Zator, Poland cellulolytic enzymes in SC. 0bliquu.s. Investigations of the cell wall (CW) of the The algae Scenedesmusobliauus var. altemas Christjuk unicellular fresh-water algae Scenedesmus and strain-633 used for isolation of CWH and CWM were bacte~olo~ica~v and algolo&ally clean. The C~~ore~~a(C~~orococc~Ze~) were performed to algae were fro& the collecti& Gf the Zootechnic determine their structure and to elaborate Institute in Zator. The culture conditions and methods of isolation of native CWH have been described possible mild methods of degradation. earlier [7]. CWM were obtained from 2 month cultures Until the present time the following CW by centrifuging at 800 g for 15 min. The white upper layer from the sediment was harvested with a spatula. degradation methods have been described: The thus-obtained CWM was cleaned by repeated (1) mechanical (a) homogenisation with glass centrifugation in a conical test-tube, diameter 1.0 cm, in conditions as described above. The CWM, after beads [l]; (b) high-pressure extrusion [2]; washing three times with water, was prepared observation by fixation in (2) ultrasonic [3, 4]; (3) biological (phages) for ei~~o~icros~opic KMnO, and OsO,, drying in aqueous solutions with r51. increasing concentration of methanol, embedding in It seemstheoretically possible to elaborate methacrylate and additionally contrasting with uranyl acetate. The method is described in detail in a previous a degradation method on the basis of knowl- work [7]. The thin sections were observed with an edge of the intact CW structure, by the use electron microscope JEM 7. of enzymes from egzogenic (lyse), or endogenie (autolyse) sources, as is done with a The results are presented in figs l-4 and textfig. I. From fig. 1 it can be concluded that number of bacteria. The only information concerning the CW structure of the algae Scenedesmus is given by Bisalputra et al. [6]. On the basis of histochemical and electronmicroscopical observations, the authors have distinguished three layers in the CW: the 3. Bwak, M L, BeGak, W & Rabello, M N, Chromosoma 19 (1966) 188. 4. Ibid 22 (1967)~192. 5. wak, M L, Denaro, L & Becak, W, Cytogenetics 9 (1970) 225. 6. Becak. M L. Becak. W & Vizotto, L D, Experie&la 26 (1970) 545.’ 7. Bwak, W, Bqak, M L, Lavalle, D & Schreiber, G, Chromosoma 23 (1967) 14. 8. Beqak, W, Schwantes, A R & Schwantes, M L B, J exutl zoo1 168 (1968) 473. 9. Bqak, W, Bqak, M L; Schreiber, G, Lavalle, D & Amorim. F 0. Exuerientia 26 (1970) 204. 10. Bqak, W & Goissis, G, Experientia. In press. 11. Ohno, S, Sex chromosomes and sex-linked genes. Springer, Berlin (1967). 12. Plagemann, P G W, Gregory, K F & WrobIewski, F, Biochem Z 334 (1961) 37.
Exptl CeN Res 63