A Sec7-related protein in Paramecium.

We have cloned and sequenced a SEC7-related gene in Paramecium tetraurelia that contains an open reading frame for 1135 amino acids encoding a 133 kDa protein, PSec7. Sec7, first identified in vesicular trafficking mutants in yeast, and its phylogenetic homologues function as guanine-nucleotide exchange factors for small G-proteins such as ARF (ADP-ribosylation factor). The deduced amino acid sequence in PSec7 for the motifs that form the ARF binding site are more than 70% identical to yeast Sec7 and similarly identical to ARNO, the human ARF exchange factor, with correct positioning of the critical glutamic acid residue within the motif region. Overall, the identity of PSec7 to yeast Sec7 is 32%. The deduced amino acid sequence also has five sequences that resemble IQ motifs, EF hand binding domains found in all myosins, and two pleckstrin homology domains. Similar sequences are present in yeast Sec7 and other Sec7-related molecules. A protein kinase A phosphorylation site may also be present. Southern blots suggest that a single gene encodes PSec7. Northern blots show that the message encoding PSec7 is induced on deciliation, followed by ciliogenesis, which suggests a role for PSec7 in cilia such as transport or targeting of ciliary membrane components.     

Erratum to Matthew Stokes et al., Cell Cycle Volume 7, Issue 14 ; pp. 2097-2099

In the paper, A wide-ranging cellular response to UV damage of DNA by Matthew P. Stokes and Michael J. Comb (Cell Cycle 2008; 7:2097-9), Table 1 was presented incorrectly. Protein classes not previously known to be phosphorylated in response to UV damage of DNA should have been shown in red. The correct table is found below.     

ATP is required for correct folding and disulfide bond formation of rotavirus VP7

Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.     

Fine-structure analysis of the P7 plasmid partition site.

The par region of bacteriophage P7 is responsible for active partition of the P7 plasmid prophage into daughter cells. The cis-acting partition site was defined precisely as a 75-bp sequence that was necessary and sufficient to promote correct segregation of an unstable vector plasmid when the two P7 partition proteins, ParA and ParB, were supplied in trans. Roughly the same region was necessary to exert partition-mediated incompatibility. The minimal site contains an integration host factor (IHF) protein binding site bracketed by regions containing heptamer repeat sequences that individually bind ParB. An additional sequence forms the left boundary of the site. Site-directed mutations in the latter sequence, as well as the IHF motif and the rightmost ParB box, blocked site function. Although the P7 site shares 55% sequence identity with its counterpart in bacteriophage P1, functional interactions between the partition sites and the Par proteins of the two plasmids were entirely species specific in vivo. The P1 sequence has similar IHF and ParB binding motifs, but the left boundary sequence differs radically and may define a point of species-specific contact with the Par proteins. No evidence was found for the existence of a functional P7 analog of the P1 parS core, a small subregion of the P1 site that, in isolation, acts as an enfeebled partition site with modified incompatibility properties.     

Cell-autonomous and -nonautonomous functions of LAR in R7 photoreceptor axon targeting.

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.     

Function of an internal bacteriophage T7 core during assembly of a T7 procapsid.

A DNA-free, proteinaceous procapsid of bacteriophage T7 (capsid I) has been shown in previous studies to consist of an external, spherical shell (envelope) and an internal, cylindrical core with fibrous projections that connect the core to the envelope. To determine the role of the core in assembly of the envelope of capsid I, the kinetics of appearance of capsid I and possible intermediates in capsid I assembly (AG particles) were determined in the presence and absence of the core. For obtaining these data, agarose gel electrophoresis was used and appeared to be a technique more accurate and efficient than techniques used for obtaining similar data in the past. The results of these experiments were: (i) in the presence of the core, AG particles behaved kinetically as intermediates in the assembly of capsid I; (ii) in the absence of the core, assembly of capsid I terminated prematurely and AG particles accumulated. These and other data have been interpreted by assuming that: AG particles are breakdown products of precursors of capsid I; these precursors have uncorrected errors in the assembly of their envelope; and a function of the core is to correct these errors.     

The O.H. 7 (Olduvai Gorge,Tanzania) hominid partial brain endocast revisited

The O.H.7 brain endocast was reexamined using a stereoplotting apparatus to quantify the amount of distortion. Some 127 measurements for each of the left and right sides were taken and treated statistically by student t-tests, both in paired and grouped fashion. Grouped data indicated no significant left-right differences. Paired data suggested three small regions of distortion, which produced three pairs of left-right differences that were significantly different. Given the very local nature of these minor distortions, it is concluded that the original reconstruction by Tobias was essentially correct. In addition, multiple regression analyses of selected chord-arc dimensions suggest that a volume in excess of 700 ml is most probable.     

Drosophila LAR regulates R1-R6 and R7 target specificity in the visual system.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye connect to specific targets in the optic lobe. Using a behavioral screen, we identified LAR, a receptor tyrosine phosphatase, as being required for R cell target specificity. In LAR mutant mosaic eyes, R1-R6 cells target to the lamina correctly, but fail to choose the correct pattern of target neurons. Although mutant R7 axons initially project to the correct layer of the medulla, they retract into inappropriate layers. Using single cell mosaics, we demonstrate that LAR controls targeting of R1-R6 and R7 in a cell-autonomous fashion. The phenotypes of LAR mutant R cells are strikingly similar to those seen in N-cadherin mutants.     

7. SHORT NOTES

Randall, R. and Ross, G. J. B. 1979. Increasing population of Cape Gannets on Bird Island, Algoa Bay, and observations on breeding success. Ostrich 50:168-175.

The South African Gannet Sula capensis on Bird Island has doubled in numbers between 1956 and 1974 and the process is continuing. The increase is similar to that of the two closely related species S. serrator and S. bassana. We estimated from the colony surface area and nest density that the number of birds with nests in 1974/5 was approximately 77 000. Making allowance for non-breeders, there were about 100 000 adults in 1974/5. Guano harvests from 1938–75 show considerable annual fluctuations with an overall downward trend. Poor agreement was found when relating guano harvests to population estimates, when the latter are gauged by colony surface area. We suggest that guano harvests are not good indicators of gannet population trends on Bird Island, primarily because of harvesting methods and environmental conditions. The number of chicks which fledged in the 1976/7 season in a section of the colony was far fewer than expected, even when allowances were made for egg-loss and chick mortality. A combination of heavier-than-usual rainfall and poor management techniques have resulted in high egg-loss and low chick numbers.     

Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-negative bacteria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribosomes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products efficiently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bacteriophage phi 29). It is concluded that strong Shine-Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional determinants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.     

The vacuole vs. the lysosome

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

Influence of the NaCl or CaCl2 concentration on the structure of heat-set bovine serum albumin gels at pH 7

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl(2)) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl(2) was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength.     

The P-SSP7 cyanophage has a linear genome with direct terminal repeats

P-SSP7 is a T7-like phage that infects the cyanobacterium Prochlorococcus MED4. MED4 is a member of the high-light-adapted Prochlorococcus ecotypes that are abundant in the surface oceans and contribute significantly to primary production. P-SSP7 has become a model system for the investigation of T7-like phages that infect Prochlorococcus. It was classified as T7-like based on genome content and organization. However, because its genome assembled as a circular molecule, it was thought to be circularly permuted and to lack the direct terminal repeats found in other T7-like phages. Here we sequenced the ends of the P-SSP7 genome and found that the genome map is linear and contains a 206 bp repeat at both genome ends. Furthermore, we found that a 728 bp region of the genome originally placed downstream of the last ORF is actually located upstream of the first ORF on the genome map. These findings suggest that P-SSP7 is likely to use the direct terminal repeats for genome replication and packaging in a similar manner to other T7-like phages. Moreover, these results highlight the importance of experimentally verifying the ends of phage genomes, and will facilitate the use of P-SSP7 as a model for the correct assembly and end determination of the many T7-like phages isolated from the marine environment that are currently being sequenced.     

Erratum to Eric Richter,et al. Epigenetics Volume 2, Issue 2; pp. 100–105

In the paper, A Role for DNA Methylation in Regulating the Growth Suppressor PMEPA1 Gene in Prostate Cancer, by Eric Richter, Katsuaki Masuda, Christopher Cook , Mathias Ehrich, Atekelt Y. Tadese, Hongyun Li, Anthony Owusu, Shiv Srivastava and Albert Dobi (Epigenetics 2007; 2:100–105), the authors found that the DNA probeset of GSTP1 is inaccurately shown (Materials and Methods, page 101). In the actual experiments, the correct set was used, thus the result and interpretation are correct.     

Isolation and Identification of 2-Methylxanthopurpurin-7-methylether from the Mycelium of Alternaria porri

An attempt was made to estimate the base composition of DNA from UV absorption spectrum of its enzymatic digest. Preheated DNA sample was denatured, dialyzed and hydrolyzed to nucleotides with nuclease P1. A half of the digest was dialyzed and absorbance of the un-dialyzed digest was read at 16 different wavelengths with the dialyzed one as a reference. The data were input to a computer with standard extinction coefficients. The G+C contents thus obtained were fairly reproducible and reliable. An easier computing with a table calculator was found to be satisfactory for a routine work. It was noted that mathematically correct program does not always give reasonable answers. Base compositions of some DNA estimated by this method were also reported.     

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.     

PHYSIOLOGICAL ONTOGENY : A. CHICKEN EMBRYOS. XIII. THE TEMPERATURE CHARACTERISTIC FOR THE CONTRACTION RATE OF THE WHOLE HEART.

The relation of heart rate frequencies to temperature in intact chicken embryos has been studied and the temperature characteristics calculated for each of a number of ages. These have been found to vary from 14,000 or better 12,000 µ for embryos 3 days old, to about 6,000 µ for others of 15 days. There appears to be a systematic change with time. If this inference is correct, important correlation with other properties of the pace-making function in the intact heart should become possible.     

Lactoperoxidase-catalyzed iodination of human IgM. Differences between 7 S IgM and 19 S IgM and between cell surface 7 S IgM and serum 7 S IgM.

We have studied the lactoperoxidase-catalyzed iodination of human IgM and have measured the ratio of radioactivity incorporated into the mu chain to that incorporated into the L chain (i.e. the mu/L ratio). Both 7 S and 19 S IgM were examined. The ratio of radioactivity was found to be larger for 7 S IgM than for 19 S IgM for all four of the monoclonal IgM proteins examined. The data suggest that some tyrosines of the mu chain which are buried and not available for iodination in 19 S IgM become exposed on conversion of 19 S IgM to 7 S IgM. The mu/L ratio for the IgM found on the cell surface of RPMI 8392 cells was significantly smaller than the ratios for all of the five 7 S IgM proteins studied in solution. It appears, therefore, that a portion of the mu chain of the cell surface IgM of the RPMI 8392 cells is buried in the membrane.