The genome of the tropical tree Theobroma cacao L.

2C values for angiosperms vary over 2500-fold and a positive correlation exists between C-value and latitude in herbaceous plants. Woody plants differ from herbaceous plants in chromosome size and C-value. In addition, tropical hardwoods have smaller chromosomes than other tropical plants and do not share the correlation of minimum generation time with genome size seen in herbaceous plants. Theobroma cacao is a tropical hardwood cultivated for its beans, which are used to make chocolate and cocoa butter. Its cytology is typical of the pantropical and subtropical family Sterculiaceae. Its small chromosomes, single secondary constriction, and lack of C-banding suggest a small genome. The genome size of T. cacao, measured by reassociation kinetics, is 2.01 x 10(8), which is small compared to both temperate and tropical plants previously studied. We also provide data on the melting point, base composition, and relative extent of methylation (at sites most commonly methylated in higher plants), of T. cacao DNA.

Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds.

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.

Pregnancy stimulates DNA synthesis in R3230AC mammary adenocarcinoma.

The transplantable R3230AC mammary adenocarcinoma was grown in virgin, pregnant and lactating rats and in vivo rates of [3H]-thymidine incorporation into DNA and acid-soluble dTTP were compared between tumor and host mammary glands. The tumor differed from the host gland in that the rates of uptake and phosphorylation of the injected thymidine remained unchanged throughout the lactation cycle, but the dTTP pool increased greatly during pregnancy and declined during lactation. In both tumor and host gland, DNA labeling rates were higher during pregnancy than during lactation. Tumor DNA synthesis rates, in terms of incorporated dTMP, increased markedly during pregnancy and returned to pre-pregnant rates following parturition and during lactation. This pattern was similar to host mammary glands, but the change was of a greater magnitude. The data illustrate the usefulness of growing a transplantable mammary tumor in rats of varying physiological states. This way, similarities and differences between a mammary tumor and the host mammary gland regarding their responses to the hormonal milieu of pregnancy and lactation can be assessed.

Ribonuclease H activity in rat mammary gland during the lactation cycle and in R3230AC mammary adenocarcinoma.

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.

Transfer RNA and aminoacyl transfer RNA in developing rats.

The total transfer RNA (tRNA) level in the liver, kidney, skeletal muscle and heart muscle of developing rats was determined by purification using (3H)tRNA as an internal standard. Liver and kidney contained almost twice as much tRNA per gram tissue as heart and skeletal muscle. There were no apparent differences between the sexes. The aminoacylation capacities of six tRNA species (alanyl, aspartyl, leucyl, methionyl, phenylalanyl, and tryptophanyl) from rat liver were not different during 3 developmental stages (suckling, weaning and young adult), and there were also no differences noted between males females. The in vivo percent aminoacylation of 4 tRNAs (aspartyl, leucyl, methionyl, and phenylalanyl) was lower during the newborn and suckling periods than in weaning and young adult rat livers. The tRNA of young adults was almost completely aminoacylated in vivo with the exception of alanyl-tRNA.

Cardiac glycoside effects on rat and guinea pig phospholipids.

The phospholipid content of adrenal glands, heart, intestinal mucosa, kidney, liver, pancreas, plasma, skeletal muscle, and spleen was compared in adult male rats and guinea pigs after intraperitoneal injection of 0.15 M NaCl (saline), 40% propylene glycol-10% ethanol (vehicle), or digoxin (rats 1 mg/kg; guinea pigs 0.1 mg/kg). In control (untreated) rats and guinea pigs total phospholipid phosphorous was twice as high in cardiac muscle as in skeletal muscle (800 vs. 400 mug/g tissue); lysophosphatidyl choline (LPC) values were similar in the two tissues (24 vs. 21 mug/g) in guinea pig; 11 vs. 15 mug/g in rat). Comparative phospholipid values between the two animals were similar in all tissues except adrenal glands (rat approximately 1200 mug/g; guinea pig approximately 1900 mug/g) and plasma (rat approximately 32 mug/ml; guinea pig approximately 11 mug/ml). In digoxin treated compared to control rats no differences between individual phosphatides were noted in any of the tissues examined whereas in guinea pigs the same comparison revealed differences in 8 instances, 4 of which were in LPC. Phospholipid levels in both species changed as a result of saline and vehicle injection.

Alkaline ribonuclease and ribonuclease inhibitor in mammary gland during the lactation cycle and in the R3230AC mammary tumour.

Alkaline RNAase (ribonuclease) and RNAase inhibitor were assayed to determine the potential role of the degradative process in regulating the amount of RNA in the mammary gland and mammary tumour. Very little free alkaline RNAase activity was found in the cytosol fraction of the mammary gland of virgin, pregnant, lactating or involuting Fischer rats. However, addition of p-chloromercuribenzoate to the assay medium revealed latent RNAase which, when expressed on a DNA basis, decreased during pregnancy and lactation. The cytosol latent RNAase is stable in 0.125 M-H2SO4. The non-cytosol RNAase activity also decreased during pregnancy and lactation. Addition of Triton X-100 produced slightly higher activity at all stages tested. The inhibitor activity in rat mammary gland was very low before pregnancy, increased gradually during pregnancy and more dramatically at parturition, continued to increase throughout lactation and returned to resting-gland values by the sixth day of involution. The increase during pregnancy may be due to the increased cellularity of the gland, whereas the gain during lactation was more than could be accounted for by increases in cell number. The R3230AC transplantable mammary tumour resembles the normal gland in early lactation with respect to both its cytosol and non-cytosol alkaline RNAase activities and its moderately high content of RNAase inhibitor. The relatively high inhibitor and low RNAase activities in both the gland of the lactating rat and in the tumour are of potential significance in maintaining high amounts of RNA and increased rates of protein synthesis in these tissues.

Increase in rat liver ribonuclease inhibitor levels during the neonatal period.

Alkaline ribonuclease (RNase) activity in nuclear, mitochondrial, and cytoplasmic fractions of rat liver is higher in newborn animals than in young adults. The level of cytoplasmic RNase inhibitor is low in newborn rat liver and increases more than two fold by 10 days of age.