The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Consequences of host plant chemical and physical variability to an associated herbivore

The effects of watering and fertilizer treatments on the vigor and biochemistry of the willow,Salix lasiolepis, and subsequent colonization and survivorship of its gallforming herbivore,Euura lasiolepsis, were investigated in two field experiments. Some plants received low (LW), intermediate (MW) or high (HW) levels of water as treatments, while others received no (OF), low (LF) or high (HF) fertilizer levels. In the watering experiment, plant protein concentrations decreased, while growth rate and number of galls per plant increased with increased water treatments. Plant growth proved to be the best correlate of sawfly attack. Sawfly survivorship increased slightly with greater watering, and phenol concentrations showed no pattern among treatments. In the fertilization experiment, leaf protein increased with fertilization, although shoot length, number of galls and survivorship ofE. lasiolepis survivorship were greatest in intermediate treatment plants. In both experiments, plant growth, rather than protein or phenol levels, was the best predictor of sawfly attack and survivorship. In a natural experiment with galls on wild plants, galled tissue had significantly greater protein concentrations and lower phenol concentrations than did ungalled tissue. We suggest that gallformers modify host plant biochemistry within willow galls, which may explain why the chemical parameters of ambient plant quality we tested were less predictive than plant growth.     

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [³⁵S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.

The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins.     

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.     

Elevated temperature alters the ionic dependence of amine-induced pacemaker activity in a conditional burster neuron

Summary The anterior burster neuron of the lobster (Panulirus interruptus) stomatogastric ganglion is a conditional burster that functions as the primary pacemaker for the pyloric motor network. When modulatory inputs to this cell are blocked, it loses its bursting properties and becomes quiescent. Applications of the monoamines, dopamine, octopamine or serotonin restore rhythmic bursting in this cell (Flamm and Harris-Warrick 1986). At 15 °C, serotonin- and octopamine-induced oscillations depend critically upon sodium entry (blocked by low sodium saline or tetrodotoxin); dopamine-induced oscillations depend upon calcium entry (blocked by reduced extracellular calcium; Harris-Warrick and Flamm 1987). We show here that the ionic dependence of amine-induced oscillations in the anterior burster cell differs at 15 and 21 °C. At 21 °C, all amines have the potential to induce rhythmic oscillations in saline containing tetrodotoxin. At the elevated temperature and in tetrodotoxin, both calcium and sodium currents are essential for the maintenance of dopamine-induced oscillaions; serotonin-induced oscillations do not depend upon either calcium or sodium alone; octopamine-induced oscillations do not depend upon calcium and show a variable dependence upon sodium. Thus, multiple ionic mechanisms, which vary with both the modulator and the ambient temperature, can be recruited to support rhythmic activity in a conditional burster neuron.Abbreviations AB anterior burster - PD pyloric dilator - PY pyloric constrictor - DA dopamine - 5HT serotonin - Oct octopamine - STG stomatogastric ganglion - TTX tetrodotoxin - GSP graded synaptic potential     

Association of polymorphisms in the HLA-B region with extended haplotypes

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLa-B, respectively. Five different RFLP variants occuring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30, DR3], [HLa-Bw62,SC33,Dr4], [HLa-B44,SC30,Dr4], and [HLA-Bw47,FC91,0DR7]. The patterns of 6.9 kb/ 3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21, DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was suprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Organization and expression of a second chromosome follicle cell gene cluster in Drosophila

Four genes expressed during the period of vitelline membrane formation are clustered within 8 kb of DNA in region 26A of the second chromosome. Temporal and quantitative difference in the profiles of accumulated RNA suggest that the genes are independently regulated although they are selectively expressed during the stages of vitelline membrane biosynthesis. In situ hybridization and S1 analyses of RNAs from fractionated eggchambers established that these genes are active only in the follicle cells. S1 mapping with in vitro synthesized RNA probes shows that three of the genes are tandemly oriented. All four appear to be intronless. In vitro translation products from hybrid-selected RNAs indicate that two of these genes code for major vitelline membrane proteins. Sequence analysis of these two genes support this conclusion. The cell- and stage-specific expression of the other two genes, encoding less abundant RNAs, suggests that they also play a role in early eggshell production.     

DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis.

An in vivo assay was used to define the DNA requirements at the bacteriophage G4 origin of complementary-strand DNA synthesis (G4 origin). This assay made use of an origin-cloning vector, mRZ1000, a defective M13 recombinant phage deleted for its natural origin of complementary-strand DNA synthesis. The minimal DNA sequence of the G4 genome sufficient for the restoration of normal M13 growth parameters was determined to be 139 bases long, located between positions 3868 and 4007. This G4-M13 construct was also found to give rise to proper initiation of complementary-strand synthesis in vitro. The cloned DNA sequence contains all the regions of potential secondary structure which have been implicated in primase-dependent replication initiation as well as additional sequence information. To address the role of one region which potentially forms a DNA secondary structure, the DNA sequence internal to the G4 origin was altered by site-directed mutagenesis. A 3-base insertion at the AvaII site as well as a 17-base deletion between the AvaI and AvaII sites both resulted in loss of origin function. The 17-base deletion was also generated within the G4 genome and found to dramatically reduce the infectious growth rate of the resulting phage. These results are discussed with respect to the role of the G4 origin as the recognition site for primase-dependent replication initiation and its possible role in stage II replication.