The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal anti-body, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.     

Oligochaeta in Spartina stems: the microdistribution of Enchytraeidae and Tubificidae in a salt marsh,Sapelo Island,USA

The distribution and abundance of Enchytraeidae and Tubificidae in and around Spartina alterniflora plants in a tidal salt marsh on Sapelo Island, Georgia, USA were studied using two different sampling techniques: wet funnel extraction and stem dissection. At least 80% of all worms inhabited leaf sheaths at the bases of S. alterniflora plants, and densities were low in sediment, root and surface debris samples. Oligochaete densities were dependent on the position within the marsh, the height on stems and the stage of sheath decay. Six predominant species were identified and included Marionina appendiculata, Marionina spartinae, Marionina waltersi, Marionina paludis, and Monopylephorus parvus. Individual species were distributed differently on stems and enchytraeids were more common than tubificids on standing-dead and further up S. alterniflora stems. Estimates of oligochaete densities in salt marsh habitats are increased dramatically when the numbers of worms on stems are considered. Possible advantages of the stem microhabitat are discussed in relation to the biology and ecology of oligochaetes.     

The evolutionary expansion of the trypanosomatid flagellates

The trypanosomatids combine a relatively uniform morphology with ability to parasitise a very diverse range of hosts including animals, plants and other protists. Along with their sister family, the biflagellate bodonids, they are set apart from other eukaryotes by distinctive organisational features, such as the kinetoplast-mitochondrion and RNA editing, isolation of glycolysis enzymes in the glycosome, use of the flagellar pocket for molecular traffic into and out of the cell, a unique method of generating cortical microtubules, and bizarre nuclear organisation. These features testify to the antiquity and isolation of the kinetoplast-bearing flagellates (Kinetoplastida). Molecular sequencing techniques (especially small subunit ribosomal RNA gene sequencing) are now radically reshaping previous ideas on the phylogeny of these organisms. The idea that the monogenetic (MG) trypanosomatids gave rise to the digenetic (DG) genera is losing ground to a view that, after the bodonids, the African trypanosomes (DG) represent the most ancient lineage, followed by Trypanosoma cruzi (DG), then Blastocrithidia (MG), Herpetomonas (MG) and Phytomonas (DG), with Leptomonas (MG), Crithidia (MG), Leishmania (DG) and Endotrypanum (DG) forming the crown of the evolutionary tree. Vast genetic distances (12% divergence) separate T. brucei and T. cruzi, while the Leishmania species are separated by very short distances (less than 1% divergence). These phylogenetic conclusions are supported by studies on RNA editing and on the nature of the parasite surface. The trypanosomatids seem to be able to adapt with ease their energy metabolism to the availability of substrates and oxygen, and this may give them the ability to institute new life cycles if host behaviour patterns allow. Sexual processes, though present in at least some trypanosomatids, may have played only a minor part in generating diversity during trypanosomatid evolution. On the other hand, the development of altruistic behaviour on the part of some life cycle stages may be a hitherto unconsidered way of maximising fitness in this group. It is concluded that, owing to organisational constraints, the trypanosomatids can undergo substantial molecular variation while registering very little in the way of morphological change.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Isolation of an Internal Deletion Mutant of the Arabidopsis thaliana ABI3 Gene

An Arabidopsis thaliana mutant that produces green seeds thatare highly insensitive to exogenous ABA, non-dormant and severelydesiccation intolerant was isolated from a population of fastneutron-irradiated seeds. Molecular and genetic analysis ofthis mutant shows that these phenotypes are caused by an internaldeletion of approximately one third of the ABI3 gene. Thereforeabi3 mutants with the above phenotypes are representative ofnull alleles at this locus. (Received December 3, 1993; Accepted January 22, 1994)     

Interaction of mithramycin with isolated GC and CG sites

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)_n and A_n · T_n using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)_n appear to be bound better than those surrounded by A_n · T_n. Although no footprints are produced at T₉GCA₉ and T₁₅CGA₁₅, DNase I cleavage is enhanced with the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)_n · (CT)_n.     

Partial sequence analysis of 130 randomly selected maize cDNA clones.

As part of a project to identify novel maize (Zea mays L. cv B73) genes functionally, we have partially sequenced 130 randomly selected clones from a maize leaf cDNA library. Data base comparisons revealed seven previously sequenced maize cDNAs and 18 cDNAs with sequence similarity to related maize genes or to genes from other organisms. One hundred five cDNAs show little or no similarity to previously sequenced genes. Our results also establish the suitability of this library for large-scale sequencing in terms of its large insert size, proper insert orientation, and low duplication rate.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham