Mimicry by cytokinin of phytochrome-regulated inhibition of chloroplast development in etiolated cucumber cotyledons

A study of the kinetics of chlorophyll (Chl) synthesis in cotyledons of etiolated cucumber seedlings ( Cucumis sativus L . cv. Delilah) treated with 5×10^-5 M -ben-zyladenine (BA) showed that cytokinin, like a red light pulse, could inhibit as well as promote pigment accumulation depending on the length of the dark period following induction. Spraying intact, dark-grown seedlings with BA, 24 h prior to white light exposure, eliminated the lag phase in Chl synthesis, while treatment with hormone 72 h before greening not only delayed the onset of synthesis, but it also reduced the amount of Chl accumulated after 24 h continuous white light. Impairment of Chl formation was correlated with inhibited regeneration of protochlorophyll and delayed appearance of the light harvesting Chl alb polypeptide. Application of σ-aminolevulinic acid (15 m M ) 2 h before white light exposure shortened the lag phase in Chl synthesis in control as well as in inhibited cotyledons, but the adverse effect of the red light and BA treatments on long-term Chl accumulation (24 h) was not reversed. Application of glutamate did not stimulate Chl production. Simultaneous treatment with hormone and red light 72 h before greening enhanced their separate inhibitory effects on Chl synthesis, but when given together 24 h prior to white light, their promotive effects on pigment accumulation were not additive.     

Impaired chlorophyll formation in etiolated cucumber cotyledons following prolonged dark incubation after red light pretreatment

Red light (R) pretreatment of etiolated cucumber seedlings ( Cucumis sativus L. var. Elem) followed by prolonged dark incubation prior to white light (WL) exposure, had an adverse effect on the greening of the cotyledons. The effect was photoreversible by far-red (FR) light. Cotyledons which were dark incubated for 24 h following the R pulse greened more rapidly when exposed to WL than did the controls, while total chlorophyll (Chl) accumulation after 24 h in the light was about the same in both. However, after 48 h post-R dark incubation greening of the treated cotyledons was delayed, and their amount of Chl which accumulated after 24 h WL was about one half of that in non-treated seedlings. As the length of the post-R dark incubation period was extended Chl production became slower, so that after 96 h post-R dark incubation the Chl level in the treated cotyledons after 24 h WL was approximately 20% of the controls. No significant differences in amounts of protochlorophyll could be detected between seedlings preilluminated with R or R followed by FR. Seedlings 4-, 5- and 6-days-old at the time of R treatment showed similar degrees of impaired Chl synthesis following prolonged post-R dark incubation.     

CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.     

Organization of shared and unshared sequences in the genomes of chicken endogenous and sarcoma viruses.

The genome of the genetically transmitted endogenous C type virus of chickens, RAV-O, is closely related to that of Rous sarcoma virus (RSV). Nevertheless, these viruses differ widely in oncogenicity and regulation by the host cell. Competitive hybridization analysis of ¹²⁵I-labeled genomic RNA demonstrated that the genome of RAV-O lacks about 35% of the sequences of nondefective RSV which formed hybrids with proviral DNA from RSV-infected cells, and that the genome of transformation-defective deletion mutants of RSV (td RSV) lacks about 15% of these sequences. Conversely, about 12% of the RAV-O sequences forming hybrids with normal chicken cell DNA were not detected in the sarcoma virus. A technique was developed to map the location of these unshared sequences by competitive hybridization. The deletion in the genome of td RSV was seen to begin at about 0.2 and to end at about 0.05 of the genome length from the 3′ end of sarcoma virus RNA, confirming the results of other laboratories using the method of mapping RNAase TI resistance of oligonucleotides. The 35% of RSV sequences missing and/or diverged in the genome of RAV-O were concentrated within 40% of the sarcoma virus genome from the 3′ end, and most of this large section did not appear to form hybrids with chicken DNA under the conditions of these experiments. A low level of hybrid formation was, however, detected between uninfected chicken cellular DNA and a small fraction of the nucleotides in the region of the td deletion. Analysis of RAV-O 3′ end fragments demonstrated that the genomic sequences of RAV-O missing in RSV were concentrated at the 3′ end of the endogenous viral genome. We conclude that the sequence differences between endogenous and sarcoma viruses are largely concentrated in specific regions of the viral genome.     

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.     

Investigating the cardiac pathology of SCO2‐mediated hypertrophic cardiomyopathy using patients induced pluripotent stem cell–derived cardiomyocytes

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)‐derived cardiomyocytes (iPSC‐CMs) from SCO2‐mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca²⁺‐overload resulting in functional derangements and arrhythmias, we investigated in SCO2‐mutated iPSC‐CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to β‐adrenergic stimulation, increased [Ca²⁺]_o and angiotensin‐II (AT‐II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC‐CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca²⁺ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC‐CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.     

Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.     

Viral DNA in bursal lymphomas induced by avian leukosis viruses.

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa of Fabricius. Viral DNA in tumors and normal tissues from infected birds were analyzed by using restriction endonucleases. Viral DNA fragments diagnostic of the exogenous ALV were easily detected in tumors, uninvolved bursal tissue, kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detect in liver. Using a restriction endonuclease (SacI) which cleaves linear unintegrated ALV DNA in a single site to define integration sites in DNA from the various tissues, we were able to detect ALV DNA only in tumor tissue. We concluded that the proviral DNA detected in the various nontumor tissue must be integrated in multiple sites. The appearance of ALV integration sites uniquely in tumors suggests that they are clonal growths. Furthermore, the data suggested the presence of a single exogenous integration site for the ALV provirus in each of six early neoplastic bursal nodules. This provirus appeared to retain the organization of EcoRI and BamHI recognition sequences present in the genome of virus used to infect the birds. The ALV integration site appeared different in each of the tumors studied. In a widespread metastatic lymphoma, multiple ALV integration sites were found as well as structural alterations in at least some copies of the ALV provirus.