Effect of cadmium on the relationship between replicative and repair DNA synthesis in synchronized CHO cells.

Repair and replicative DNA synthesis were measured at different stages of the cell cycle in control and cadmium-treated Chinese hamster ovary (CHO-K1) cells. Cells were synchronized by counterflow centrifugal elutriation. Elutriation resulted in five repair and four replication subphases. On Cd treatment, repair synthesis was elevated in certain subphases. Replicative subphases were suppressed by Cd treatment, with some of the peaks almost invisible. The number of spontaneous strand breaks measured by random oligonucleotide primed synthesis assay showed a cell-cycle-dependent fluctuation in control cells and was greatly increased after Cd treatment throughout the S phase. Elevated levels of the oxidative DNA damage product, 8-oxodeoxyguanosine, were observed after Cd treatment, with the highest level in early S phase, which gradually declined as damaged cells progressed through the cell cycle.     

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.     

Differential effects of sulphur mustard on S-phase cells of primary fibroblast cultures from Syrian hamsters

Sulphur mustard (SM) (5 × 10⁻⁸ M) given to primary Syrian hamster fibroblasts growing short-term in vitro products a very sharp peak of chromatid aberrations 12–16 h after treatment.

The composition of this peak has been investigated in relation to the cell cycle using the facility to divide S-phase into 5 cytologically defined sub-phases on the basis of replication band patterns following bromodeoxyuridine incorporation. It is shown that: (1) Considerable delay and perturbation of the cycle is present. (2) A major contribution to the aberration peak comes from pre-S cells, and the highest aberration frequencies are observed in such cells. (3) The bulk of contribution from S-cells comes from the last subphase, SkV. (4) The majority of early S cells (sub-phases SkI–IV) fail to reach division within 36 h. Of the total S-cells scored, 54% are non-SkV controls but only 14% after SM. (5) Aberrations are localized to late-replicating regions in SkV but are random in pre-S. (6) No measurable perturbation of replication programme was found in chromosome arms synthesizing in late SkV after SM. (7) The rapid fall in aberration frequency at later sampling times is consistent with a quick and efficient repair of DNA lesions which is known to occur after SM alkylation.

The preferential loss of early S cells seems most likely to result from selective lethality, though differential delay and perturbation may make a contribution. It is interesting to note that the subphases missing are just those where the euchromatin replicates (early replicating R and G bands). The late-replicating chromatin, some of which is known to be dispensable in Syrian hamster, is confined to SkV, the sub-phase which appears to survive this dose of SM.     

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagle's minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.     

The relative contribution of adduct blockage and DNA repair on template utilization during replication of 1,N2-propanodeoxyguanosine and pyrimido

The role of replication blockage by the exocyclic DNA adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M1G) was determined through the use of site-specifically adducted M13MB102 genomes containing a C:C-mismatch approximately 3000 base-pairs from the site of adduct incorporation. Genomes containing either dG, PdG, or M1G positioned at site 6256 of the (-)-strand were transformed into repair-proficient and repair-deficient Escherichia coli strains and the percent template utilization was determined by hybridization analysis. Unmodified genomes containing a C:C-mismatch resulted in a percent template utilization of approximately 60 and 40% for the (-)- and (+)-strands, respectively. Transformation of PdG- or M(1)G-adducted genomes resulted in approximately a 60-40% and 50-50% (-)-strand to (+)-strand ratio, respectively, indicating that PdG and M(1)G are negligible blocks to replication in repair-proficient E. coli. This is in contrast to previous studies using (PdG:T)- and (M1G:T)-mismatched M13MB102 genomes, which resulted in a majority of the replication events using the unadducted (+)-strand and suggested that both adducts were significant blocks to replication [J. Biol. Chem. 272 (1997) 11434; Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8652]. The C:C-mismatch results, though, indicate that the large strand bias detected in the earlier studies is due to repair of the adducts and resynthesis of the (-)-strand using the (+)-strand as a template for repair synthesis. Transformation of adducted C:C-mismatched genomes into E. coli strains deficient in nucleotide excision repair did result in an increased strand bias with only approximately 20 and 34% of the replication events using the (-)-strand for PdG- and M1G-adducted genomes, respectively. The increased strand bias indicates the importance of nucleotide excision repair in the removal of PdG and M1G.     

Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21-C13 cells by the addition of serum

BHK21/C13 cells placed in medium containing low (1%) serum ceased DNA synthesis within 4 days. DNA synthesis recommenced 10 h after the readdition of serum (to 10%) to cells incubated for 6 days in serum-depleted medium. Two peaks of thymidine incorporation were observed at 12–13 h and 15–17 h, followed by a single peak of dividing cells at 25 h. The two peaks of incorporation represent variation in the extent of DNA replication during a single synchronous S phase.Uridine, deoxyadenosine and deoxyguanosine kinase activities did not decline in serum-depleted cells and, after the addition of serum, their activities showed cyclical variation about a mean involving two-fold changes in enzyme specific activity. All other enzyme activities examined were markedly decreased in resting cells.Ornithine decarboxylase activity increased 15-fold within 5 h of serum addition, but had returned to the resting level by 8 h. There was no apparent correlation between this alteration of enzyme activity and the rate of RNA synthesis.DNA polymerase, thymidine kinase and deoxycytidine kinase activities all decreased further within 4 h of the addition of serum, followed by several-fold increases in activity. The peak of DNA polymerase activity corresponded to, and encompassed, both peaks of DNA synthesis. However, thymidine and deoxycytidine kinase activities, although exhibiting two activity maxima corresponding to the peaks of DNA synthesis, were at their highest levels in G2.     

Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

Detailed cell cycle analysis in human lymphocytes; Application to γ-irradiated cells

Summary A method based on BrdU incorporation for analyzing in detail the kinetics of the cell cycle is described. The S phase has been subdivided into five subphases, each recognizable by their BrdU incorporation pattern at metaphase. The method can be useful for the study of abnormal cell cycles, and may have particular application in mutagenesis studies concerning the various subphases of the S phase, without using synchronization techniques. An application of the method is described, showing that -irradiation, during the course of the S phase, leads to a lack of cells which were in early S phase at the time of irradiation. This finding can be related either to a higher lethality at this stage of the cell cycle or to a delay in completion of DNA replication after irradiation.Hoider of a C.E.C. scholarship     

Influence of the incubation temperature on the autolytic activity of Lactobacillus acidophilus

M.L. FERNANDEZ MURGA, A. PESCE DE RUIZ HOLGADO AND G.F. DE VALDEZ, 1995. The effect of temperature and growth phase on the autolysis of Lactobacillus acidophilus CRL 640 was studied. The maximal rate of autolytic activity ( ca 48% cell lysis) was found at 45°C. At this temperature, two peaks were detected: the first one at the early exponential phase of growth and the second lysis peak during the transition stage from the exponential to the stationary phase. The release of intracellular compounds absorbing at 260 and 280 nm was also detected at 45°C. The microscopic observations revealed morphological changes and the presence of ghost cells. At 37°C, the low autolytic activity obtained would be related to the normal cell cycle of growth.     

Structural Contribution of the Carbohydrate Moiety to the Alkaline Hydrolysis of Aryl Glycosylamine Linkages

Differential scanning calorimetry (DSC) thermograms of soybean protein isolate developed two peaks corresponded to 11S and 7S globulin, the denaturation temperatures of which were 93.3 and 76.5°C, respectively, with 94% water. These peaks shifted to higher temperatures with lower water contents of the sample. At 47% water, there were two peaks, at 149 and 118.7°C, and at 11% water, there was one peak at 180°C. The DSC thermogram measured during cooling and reheating gave no peak. The soybean protein isolate was heated with 24.5% water at 100°C and then mixed with more water to the water contents of 94%. This sample gave two peaks at temperatures close to those of the original soybean protein, indicating that the soybean protein was not denatured at temperatures even above 100°C when the water content was low.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins, pigment system II particles and chlorophyll a in diethylether solution by the curve-fitting method.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.     

Monitoring of activity dynamics of an anaerobic digester bacterial community using 16S rRNA polymerase chain reaction--single-strand conformation polymorphism analysis

The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a culture-independent approach based on single-strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the digester operating at steady state, the 16S rDNA SSCP patterns of the bacterial community showed eight peaks, whereas the 16S rRNA patterns showed six peaks with a very prominent one corresponding to a Spirochaetes-related bacterium. An acidic shock at pH 6 caused an increase in the 16S rRNA level of two Clostridium-related bacteria. After a 1 week starvation period, the major bacteria present reverted to a basal 16S rRNA level proportional to their 16S rDNA level. Starvation revealed the presence of a previously undetected peak whose corresponding sequence was deeply branched into the low G+C Gram-positive bacteria phylum. Twenty-four hours after a spiked addition to the starved digester community of starch, glucose, lactate or sulphate, an upsurge in several new 16S rRNA-derived peaks was observed. Thus, the perturbation approach combined with 16S rRNA analysis revealed bacteria that had not been detected through 16S rDNA analysis.     

Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.     

BRCA1 is required for postreplication repair after UV-induced DNA damage

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent but nucleotide excision repair (NER)-independent manner. More specifically, at UV-stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and postreplicative repair. These BRCA1 functions differ from those required for DSBR.     

Small molecular weight GTP-binding proteins in human erythrocyte ghosts

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.     

Effects of DNA replication inhibitors on UV excision repair in synchronised human cells.

When HeLa cells are irradiated with UV and treated with the DNA synthesis inhibitors hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), DNA strand breaks accumulate at sites where excision repair of DNA damage has been inhibited after the incision step. This break accumulation occurs in mitotic, G1 and S phase cells. But UV-induced repair synthesis of DNA, as measured by [3H]thymidine incorporation into unreplicated DNA, is not inhibited by HU and ara C in G1 or S phase cells, even though replicative synthesis is virtually abolished. Repair and replication must therefore utilise different DNA precursor pools, or different DNA synthetic systems; and the action of Hu and ara C in causing strand break accumulation may occur at the ligation step of excision repair.     

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.Subject terms: Cancer, DNA damage and repair     

The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.     

Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells.

Replication of the oxidative lesion 8-oxo-7,8-dihydroguanine (GO) leads to the formation of both 8-oxo-7,8-dihydroguanine:adenine (GO:A) and 8-oxo-7,8-di-hydroguanine:cytosine (GO:C) pairs. The repair and mutagenic potency of these two kinds of base pairs were studied in simian COS7 and human MRC5V1 cells using the shuttle vector technology. Shuttle vectors carrying a unique GO residue opposite either a C or an A were constructed, then transfected into recipient mammalian cells. DNA repair resulting in G:C pairs and mutation frequency, were determined using resistance to digestion by the Ngo MI restriction enzyme for screening and DNA sequencing of suspect mutants. Results showed that the GO:C mismatch was well repaired since almost no mutations were detected in the plasmid progeny obtained 72 h after cell transfection. The GO:A pair was poorly repaired since only 32-34% of the plasmid progeny contained G:C whereas two thirds contained A:T at the original site. Repair kinetics measured with a non-replicating vector deleted by 13 bp at the SV40 replication origin, showed that GO:A was slowly repaired. Only 30% of the mispairs were corrected in 12 h. During this time 100% of the plasmids containing GO:A pairs were replicated as seen by the replication kinetics in a vector with an intact SV40 replication origin. These results show that, under our experimental conditions, replication is occurring before completion of DNA repair which explains the high mutagenic potency of the GO:A mispair.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.