The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Geographic variation in viral load among hepatitis B carriers with differing risks of hepatocellular carcinoma.

The risk of hepatocellular carcinoma (HCC) varies significantly among hepatitis B virus (HBV) carriers from different geographic regions. We compared serological markers of HBV infection in adult male carriers from Haimen City, China and Senegal, West Africa, where the prevalence of chronic infection is similar. HCC mortality among HBV carriers is much higher in Haimen City than it is in Senegal (age-standardized rate, 878 versus 68 per l0(5) person-years). A dramatic difference was observed when HBV DNA levels in serum were assessed among carriers by Southern blot. In the Senegalese group (n = 289), 14.5% were HBV DNA positive by Southern blot in their 20s, and this percentage declined in each subsequent decade of age to 3.3, 2.9, and 0% thereafter. In the Chinese group (n = 285), a higher prevalence of HBV DNA positivity and a less consistent reduction were seen; 29.4% were positive in their 20s, and 30.2, 23.6, and 20.6%, respectively, were positive in each subsequent decade of age. Among 102 male Asian-American HBV carriers, the prevalence of HBV DNA positivity was intermediate between the Chinese and Senegalese populations (36.8, 10.7, 3.0, and 4.6% in each subsequent decade of age). Viral titers were similar among those who were HBV DNA positive in all three populations [median value, 10(7) virions/ml (range, 10(6)-10(9) virions/ml)]. The presence of HBV DNA in serum was positively associated with serum glutathione S-transferase, a marker of liver damage. These findings suggest that the more prolonged maintenance of productive virus infection in the Chinese carriers compared with the Senegalese carriers may explain their higher risk of HCC. This profound difference in the natural history of chronic infection may be due to earlier age of infection in China or to as yet unknown environmental or genetic factors.

Characterization of the antiviral effects of 2' carbodeoxyguanosine in ducks chronically infected with duck hepatitis B virus.

This study was carried out to evaluate benefits and limitations of long-term therapy of hepatitis B virus infections with a nucleoside analog inhibitor of virus replication. The model we used was the domestic duck chronically infected with duck hepatitis B virus by in ovo infection. 2' Carbodeoxyguanosine was used as an inhibitor of viral DNA synthesis. In all animals examined there was a reduction in virus production during therapy. A dose of 2' carbodeoxyguanosine of 10 micrograms/kg every other day reduced the number of infected hepatocytes from greater than 95% to 25% to 50% in less than 3 mo, whereas a 10-fold higher dose produced a decline to less than 10%. Histological evaluation revealed mild to moderate liver injury in ducks receiving the higher dose of 2' carbodeoxyguanosine, suggesting that disappearance of infected hepatocytes may have been accelerated by a toxic effect of the drug. Drug treatment did not completely eliminate duck hepatitis B virus from any duck, and replication was restored in all hepatocytes within a few weeks to several months after antiviral therapy was terminated. Our results suggest that elimination of a chronic infection with a single inhibitor of replication may be difficult in a host that lacks an antiviral immune response capable of eliminating at least a portion of the infected hepatocytes and of ultimately producing antibodies capable of neutralizing residual virus.

Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.

Humoral immune responsiveness in duck hepatitis B virus-infected ducks.

Immunofluorescence assays with fixed tissue sections were used to characterize antibody reactivity in sera obtained from duck hepatitis B virus-infected ducks. Under conditions of experimental infection, antibody to core antigen but not to surface antigen was detectable. A majority of the ducks infected at 8 days after hatching and a minority of those infected at 1 day after hatching showed a transient anti-core antigen humoral response; this response was stronger in the antibody-positive ducks infected on day 8 than in those infected on day 1. Antiviral antibody was not detected in the sera of ducks congenitally infected with duck hepatitis B virus. Several of the infected ducks, but none of the uninfected ducks, exhibited autoantibody reactivity for alpha-islet-cell-associated antigen.

Viral antigen expression in the pancreas of DHBV-infected embryos and young ducks.

The time course of appearance of viral antigen-positive pancreatic cells was examined in both congenitally duck hepatitis B virus (DHBV)-infected duck embryos and experimentally DHBV-infected posthatch ducks. In the embryos, the earliest detectable viral antigen-positive pancreatic cells were localized to islets and identifiable as endocrine on the basis of hormone expression. Non-islet-associated, viral antigen-positive cells appeared at a late stage of embryogenesis, following the onset of chymotrypsinogen production by exocrine tissue; a number of these viral antigen-positive cells were directly identifiable as exocrine on the basis of chymotrypsinogen expression. By contrast, in the pancreas of experimentally infected posthatch ducks, the appearance of viral antigen-positive exocrine cells (chymotrypsinogen-positive) predated the appearance of antigen-positive islet cells. These results are consistent with the possibility that viral antigen expression in exocrine tissue is dependent on the state of cell maturation.

Sequence of events in natural infection of Pekin duck embryos with duck hepatitis B virus.

The major mode of natural infection of duck hepatitis B virus (DHBV) in Pekin ducks is vertical transmission, with 95 to 100% of the embryos from DHBV-infected dams eventually becoming infected. Maternally transmitted virus is present in large quantities in the yolk of unincubated eggs and is taken up by the embryo during early development. Synthesis of DHBV DNA in the embryo begins at about 6 days of incubation and coincides with the formation of the liver. DHBV DNA synthesis is incomplete, however, until 8 to 10 days of incubation, as shown by comparing the electrophoretic patterns of DHBV-specific nucleic acid species from embryonic livers at successive stages of development. From 8 days of incubation and continuing throughout embryonic development, subviral particles, which resemble viral replication intermediates isolated from infected livers of post-hatch ducklings, appear in the circulation. These particles contain a polymerase activity that utilizes an RNA template to synthesize viral DNA. Our results suggest that certain host functions, which appear during embryonic development, may be required for DHBV replication and assembly. It is possible that in mammals a similar developmental process occurs. The failure to find human hepatitis B virus in the circulation of most babies, born to hepatitis B virus carrier women, in the first few weeks after birth may reflect such a process.

Duck hepatitis B virus and liver diseases.

The presence of duck hepatitis B virus in serum was studied in 61 ducks (24 from Chi-tung county, China, 20 from Changchun, China, and 17 from Chiba, Japan) with relation to liver disease. None of the 37 ducks from Chiba and Changchun was positive for duck hepatitis B virus as assayed by electron microscopy, endogenous deoxyribonucleic acid-polymerase activity, and hybridization with duck hepatitis B virus deoxyribonucleic acid. No liver disease was seen in these ducks. In contrast, viruslike particles were present in the serum of 12 of 24 (50%) ducks from Chi-tung, China. The presence of duck hepatitis B virus in serum was indicated by the hybridization spot test and deoxyribonucleic acid-polymerase activities. A variety of liver diseases including chronic hepatitis and cirrhosis were seen in the livers of a majority of the ducks from Chi-tung. One duck hepatitis B virus-positive duck had multicentric hepatocellular carcinoma with underlying cirrhosis. Comparison of serum duck hepatitis B virus markers and liver disease in the affected flock revealed a tendency for seronegative ducks to have advanced liver diseases. Duck hepatitis B virus infection may be used as an experimental model to test various hypotheses concerning the pathogenesis of hepatitis B virus-associated liver disease in humans.

Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus.

We tested the hypothesis that duck hepatitis B virus (DHBV) is a naturally occurring congenital infection of Pekin duck embryos. Of 219 embryos, 5-25 days after being laid, sera from 30 were found to be positive for endogenous DNA polymerase activity characteristic of hepatitis B-related viruses. The presence of the duck virus was confirmed by hybridization with cloned DHBV DNA. Viral DNA was also found in the livers of embryos incubated for 12 or 18 days. Electrophoretically different forms of DHBV DNA were identified in liver extracts that were not present in serum. These additional liver forms probably represent viral replication intermediates. These observations suggest that the vertical route is a major pathway of DHBV transmission and that viral replication may be initiated by the 12th day of embryonic life.

Immunochemistry of conjugates prepared from serum albumins and acridine nitrogen mustards (ICR mutagens).

Antibodies elicited in rabbits by immunization with conjugates prepared from serum albumins and nitrogen mustard derivatives of quinacrine (atebrin) were found to have strong binding sites complementary to the quinacrine hapten. The characteristic absorption spectrum of quinacrine made possible accurate determinations of the antigen-antibody composition of the serological precipitates. Conclusive evidence that such antibodies, in addition to reacting with the quinacrine component of heterologous protein test conjugates, bind quinacrine itself, as well as closely related acridine haptens, was provided by quantitative inhibition studies. Atebrin and the hydroxy precursors of several heterocyclic nitrogen mustards caused more than a 50% inhibition of the antigen-antibody reactions. The antibodies elicited by the quinacrine-protein conjugates in ascites tumor-bearing mice substantially neutralized the antitumor effectiveness of the low dosages (0.5 to 2.0 mumol/kg) of the acridine nitrogen mustards that were required for a demonstration of chemotherapeutic activity. In contrast, nitrogen mustard, which has no quinacrine moiety, was not affected. Immunization with unaltered serum albumin had no influence on the activity of the acridine nitrogen mustards. Quantitative in vitro inhibition studies allowed satisfactory predictions in vivo immunological reactivity.

Association of e antigen with Dane particle DNA in sera from asymptomatic carriers of hepatitis B surface antigen.

Sera containing hepatitis B surface antigen from 30 asymptomatic blood donors were assayed for e antigen (HBeAg) and antibody to e antigen (anti-HBe) by rheophoresis,. Fourteen samples (47%) had detectable HBeAg, ten (33%) had anti-HBe, and six (20%) had neither. DNA was extracted from 26 of these sera and assayed for its ability to anneal to a [32P]-DNA probe that is a copy of Dane particle DNA. All 10 HBeAg-positive samples tested contained DNA that formed specific hybrids with the DNA probe, as did one of 10 anti HBe-positive samples. Hybridization was not detected in nine sera containing anti-HBe and six sera without HBeAg or anti-HBe. Because the Dane particle is thought to be the hepatitis B virus, this association between HBeAg positivity and Dane particle DNA strongly supports the hypothesis that e antigen is a marker of the presence of the virus and, consequently, potential infectivity.

Hepatitis B surface antigen: regional variation in sub-type ratio in the Canadian Red Cross donor population.

A total of 344 sera positive for hepatitis B surface antigen from volunteer blood donors at several Canadian Red Cross centres were subtyped for ad and ay specificity by counterelectrophoresis. Of the 50 sera from Toronto 21 (42%) were ad and 29 (58%) were ay; of the 95 from Montreal 82 (86%) were ad and 13 (14%) were ay; of the 199 from Quebec 179 (90%) were ad and only 20 (10%) were ay. The w and r specificities were also determined in 125 of the samples: 123 were w; the 2 samples of r specificity were from Toronto. On the other hand, among 45 sera from patients with acute hepatitis type B in Quebec 13 (29%) were ad and 33 (71%) ay.