Effect of Benzyladenine on DNA, RNA, Protein, and Chlorophyll Contents in Intact Bean Leaves: Differential Responses to Benzyladenine According to Leaf Age

Benzyladenine (BA) was applied to intact bean (Phaseolus vulgaris) leaves at different stages of their growth. Changes in the amounts of cellular constituents resulting from the different treatments were followed and compared. RNA, protein, and chlorophyll contents, dry weight, fresh weight, and leaf area per single leaf continued increasing when leaves were treated with BA from an early stage, whereas in untreated leaves all these values levelled off or declined with advancing age. Besides these changes, BA treatment induced an increase in the DNA content. Changes in RNA content was more remarkable in response to application or deprival of BA treatment than the corresponding ones in protein and chlorophyll contents. The pattern of response to BA varied greatly according to the age at which the leaf received the treatment. As leaves aged, they lost the ability to increase their area and fresh weight in response to BA. However, continuous treatment with BA from an early stage kept the leaves young and able to respond.     

RNA SYNTHESIS IN CULTURED CHICK EMBRYO CELLS IN GROWING AND CONFLUENT PHASES

RNA synthesis at the growing phase in monolayer cultures of chick embryo fibroblasts was compared with that at confluent phases by zonal sedimentation, base composition and hybridization experiments. The nuclei were isolated by treatment with Nonidet p-40. The ratio of RNA/DNA in isolated nuclei was higher at the growing phase than that of confluent. The rate of RNA synthesis was reduced in the cells at confluent phase to 15.1% of that at the growing phase. The sucrose density gradient sedimentation pattern of nuclear RNA was on the whole the same in both phases. According to the distribution of ¹⁴C-uridine incorporated into nuclear RNA, 45S ribosomal precursor RNA was more distinct for the growing cell, while the radioactivities were found to be polydispersed, including the RNA which sedimented faster than 28S RNA in the cells at confluent phase. The base compositions and hybridization analyses indicated that ribosomal RNA was synthesized more actively in the growing cells. About 50% of newly synthesized RNA was ribosomal in the growing cells but 35% in the confluent.
It was found that newly synthesized 18S and 28S ribosomal RNAs appeared in cytoplasm after 21 and 33 min lag periods respectively. These times were exactly same in both growing and confluent phases.     

Avoidance of egg parasitism through submerged oviposition by tandem pairs in the water strider, Aquarius paludum insularis (Heteroptera: Gerridae)

Abstract.  1. Females of the water strider Aquarius paludum insularis (Motschulsky) (Heteroptera: Gerridae) carry males on their backs and oviposit under water after copulation. This study focuses on the benefit  A. paludum insularis receives by ovipositing in tandem.
2. Males guarded females in tandem through to the end of oviposition in 85% of copulations. Females in tandem dived deeper than single females, and the density of A. paludum insularis eggs increased with water depth. The proportion of eggs parasitized by a scelionid wasp, Tiphodytes gerriphagus Marchal (Hymenoptera: Scelionidae) decreased with increasing water depth.
3. These results suggest that during oviposition guarding by males is beneficial for females, because it enables pairs to dive and lay eggs deeper and in oviposition sites where the risk of egg parasitism is lower.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

CHANGES IN THE RESPONSE OF CARROT ROOT CALLUSES TO THE ROOT EXTRACT OCCURRING DURING THEIR SUCCESSIVE CULTURES

1. Alcohol extract of carrot root promoted the growth of the carrotroot callus which had been succesively cultured for more than18 months (CCL) on the medium containing WHITE'S inorganic salts,sucrose, yeast extract and 2, 4-D, but only a weak promotionwas observed for the growth of the carrot root callus whichhad been cultured for less than 14 months (CCS).
2. The activesubstances were fractionated by Amberlite IR-120and AmberliteIRA-400 into four fractions; C, D, E, and F. Eachfraction seemedto act synergistically to produce the effectof the whole carrotroot extract on the growth of CCL.
3. Fraction F of the carrotroot extract, which was adsorbed byAmberlite IRA-400 but notby Amberlite IR-120, promoted thegrowth of CCL in the presenceof other fractions, but had noeffect on the growth of CCS.So the different responses to thealcohol extract of the carrotroot calluses having differentlengths of successive cultureperiod seemed to depend mainlyon the ability of respondingto fraction F.
4. Using four strains of carrot root callusesof different origin,it was ascertained that different responsesof carrot root callusesto fraction F depended on the lengthof their culture and noton their strain-specific characters.
5. The substances active for the growth of CCL in the carrotrootextract passed through a dialysis membrane. These substanceswere little affected by autoclaving and remained in the aqueouslayer when shaken with several organic solvents: n-butanol,ethyl acetate, chloroform, benzene, ethyl ether and carbon tetrachloride.
6. Alcohol extract of carrot root also promoted the growth ofcarrotroot explant, tobacco stem callus and sunflower crowngall tissue.

(Received December 24, 1964; )     

CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

The Effect of Benzyladenine on Changes in Nuclease and Protease Activities in Intact Bean Leaves during Ageing

Primary leaves of intact bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages during growth. Changes in DNase, RNase, and proteas activities in the leaves were followed. Unlike the case of various excised tissues, cytokinin raised the activities of these hydrolases in intact bean leaves. Because BA elevated the levels of DNA, RNA, and protein in intact leaves, it may stimulate both synthesis and decomposition of these cellular constituents. The hydrolase activities showed differential responses to BA according to the age at which the leaf received the hormone treatment.