Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

Inheritance of Resistance to Crown Gall in Pisum sativum

We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10⁹ bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three `supersusceptible' genotypes, which developed very large tumors, also were identified.     

The effects of light and temperature on photosynthate partitioning in Antarctic freshwater phytoplankton

The effects of temperature and radiation flux on the partitioningof photosynthetically fixed carbon into four intracellulai metabolicpools was investigated for natural phytoplankton assemblagesfrom an Antarctic freshwater lake. At ambient temperature, proteinsynthesis was saturated at low photon flux densities (30–40µmol m^–2 s^–1) and above this flux fixed carbonwas increasingly stored as lipid and polysaccharide. Increasingtemperature raised both the saturated rate of protein synthesisand the photon flux at which saturation occurred. There wasa corresponding decline in the accumulation of reserve products,particularly at low radiation fluxes. The consequences of thispattern of uptake for the phytoplankton is discussed.     

Insights into the secretory pathway and vesicular transport in plant cells

Summary— The vectorial transport of membrane and macromolecules within the cytoplasm of eukaryotic cells has been the subject of intense investigation over the last decade. In this paper we review some of the recent advances made in our understanding of vesicle transport and the secretory pathway in plant cells.     

Reversible dissociation of the plant golgi apparatus by brefeldin A

Summary— The effects of the drug Brefeldin A, shown to block the translocation of proteins between the endoplasmic reticulum and the Golgi apparatus in animal cells, were studied on different plant cell systems. In suspension culture cells and root tissues, the Golgi aparatus was affected by Brefeldin A treatments resulting in distortion and dissociation of the Golgi stacks, coupled with appearance of numerous vesicles in the cytoplasm. This process was reversible. Therefore, Brefeldin A provides a powerful tool with which to study Golgi dynamics and function in plant as well as in animal cells.     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L     

The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER

Proteins are co-translationally transferred into the endo-plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells ( Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.