Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation

For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal (“afucosylation”). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase.

The lpxB gene of Escherichia coli, believed to be the structural gene for lipid A disaccharide synthase, is located in the min 4 region of the chromosome. It is adjacent to and clockwise of the lpxA gene, which is thought to encode UDP-N-acetylglucosamine acyltransferase. Preliminary evidence suggests that lpxA and lpxB are cotranscribed in the clockwise direction and thus constitute part of a previously unknown operon (D. N. Crowell, M. S. Anderson, and C. R. H. Raetz, J. Bacteriol. 168:152-159, 1986). We now report the complete nucleotide sequence of a 1,522-base-pair PvuII-HincII fragment known to carry the lpxB gene. This sequence contained an open reading frame of 1,149 base pairs, in agreement with the predicted size, location, and orientation of lpxB. There was a second open reading frame 5' to, and in the same orientation as, lpxB that corresponded to lpxA. The ochre codon terminating lpxA was shown to overlap the methionine codon identified as the initiation codon for lpxB, suggesting that these genes are cotranscribed and translationally coupled. A third open reading frame was also shown to begin at the 3' end of lpxB with analogous overlap between the opal codon terminating lpxB and the methionine codon that putatively initiates translation downstream of lpxB in the clockwise direction. These results argue that at least three genes constitute a translationally coupled operon in the min 4 region of the E. coli chromosome. The accompanying paper by Tomasiewicz and McHenry (J. Bacteriol. 169:5735-5744, 1987) presents 4.35 kilobases of DNA sequence, beginning at the 3' end of lpxB, and argues that dnaE and several other open reading frames may be members of this operon.     

Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli.

Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide. Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase. Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene. We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L. Clarke and J. Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E. coli chromosome. Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase. Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.     

Nucleotide sequence analysis of the lemur beta-globin gene family: evidence for major rate fluctuations in globin polypeptide evolution

Lemur beta-related globin genes have been isolated and sequenced. Orthology of prosimian and human epsilon-, gamma-, and beta-related globin genes was established by dot-matrix analysis. All of these lemur globin genes potentially encode functional beta-related globin polypeptides, though precisely when the gamma-globin gene is expressed remains unknown. The organization of the 18-kb brown lemur beta-globin gene cluster (5' epsilon-gamma-[psi eta-delta]-beta 3') is consistent with its evolution by contraction via unequal crossing-over from the putative ancestral mammalian beta-globin gene cluster (5' epsilon-gamma- eta-delta-beta 3'). The dwarf lemur nonadult globin genes are arranged as in the brown lemur. Similar levels of synonymous (silent) nucleotide substitutions and noncoding DNA sequence differences have accumulated between species in all of these genes, suggesting a uniform rate of noncoding DNA divergence throughout primate beta-globin gene clusters. These differences are comparable with those observed in the nonfunctional psi eta pseudogene and have therefore accumulated at the presumably maximal neutral rate. In contrast, nonsynonymous (replacement) nucleotide substitutions show a significant heterogeneity in distribution for both the same gene in different lineages and different genes in the same lineage. These major fluctuations in replacement but not silent substitution rates cannot be attributed to changes in mutation rate, suggesting that changes in the rate of globin polypeptide evolution in primates is not governed solely by variable mutation rates.      

The physiological basis of seed dormancy in Avena fatua. II. On the involvement of alternative respiration in the stimulation of germination by sodium azide

Chromatography on DEAE-cellulose of an extract from etiolated leaves of sorghum ( Sorghum vulgare Pers. cv. INRA 450), a C₄ plant, gave only one form of phosphoenol pyruvate carboxylase with functional and regulatory properties of a C₃ type plant enzyme. Greening of the leaves resulted in a significant increase in activity. This increase was due to the appearance of a new form of the enzyme, which eluted at lower ionic strength and exhibited new properties. This form was glucose-6-P activated and showed a sigmoidal curve response to the concentration of the substrate phosphoerralpyruvate. These kinetic properties are typical of a C₄ plant enzyme.