Cloning of segment polarity gene homologues from the unsegmented brachiopod Terebratulina retusa (Linnaeus).

We have used the polymerase chain reaction (PCR) to amplify, clone and sequence homologues of the Drosophila segment polarity genes engrailed (en), cubitus interruptus Dominant (ciD) and wingless (wg) from the genome of the brachiopod, Terebratulina retusa (Linnaeus). The deduced translation products of brachiopod en and ciD share high levels of sequence identity with their Drosophila homologues. The brachiopod wg-related clone is divergent from Drosophila wg, although clearly a member of the wg/Wnt gene family. These results indicate that structural diversity of Drosophila segment polarity genes has been evolutionarily conserved in a divergent, ancient and unsegmented animal phylum.     

The molecular evolution of ZFY-related genes in birds and mammals

Summary We report the isolation and nucleotide sequence determination of clones derived from five ZFY-related zinc-finger genes from birds and mammals. These sequences are analyzed with reference to the previously published human genes, ZFX and ZFY, and mouse genes, Zfx, Zfa, Zfy-1, and Zfy-2. The analysis indicates that ZFY-related genes are highly conserved in birds and mammals, and that the rate of nucleotide substitution in the Y-linked genes is not as high as predicted. However, the mouse Zfy-1 and Zfy-2 genes are markedly divergent members of the ZFY gene family; we suggest this relates to X-inactivation of the mouse gene Zfx.     

Isolation, immunolocalization, and sperm-association of three proteins of 18, 25, and 29 kilodaltons secreted by the mouse epididymis.

Three murine epididymal secretory proteins have been characterized by their site of synthesis, sperm association, and tissue localization by use of polyclonal antisera and immunochemistry. Mouse epididymal protein 7 (MEP 7) was localized initially within the supranuclear regions of some principal epithelial cells in the proximal corpus while other cells remained unstained. In the mid-proximal corpus, all principal cells and stereocilia were stained, and luminal staining increased from corpus to cauda. Some clear cells in the distal corpus and cauda also showed immunoperoxidase staining. Sequential extraction of caudal spermatozoa indicated that MEP 7 was predominantly loosely associated with spermatozoa and that only a small amount of MEP 7 required detergent to extract it from spermatozoa. Examination of other rodent caudal fluids revealed a related protein in rat caudal fluid of 32 kDa, and amino acid sequence analysis of MEP 7 showed a 68% sequence similarity with rat proteins AEG and D/E. MEP 9 immunolocalized within the cytoplasm of all principal cells of the distal caput. In a transition zone between the distal caput and the corpus, some principal cells were stained while others were not. Distal to the corpus, the principal cell staining gradually decreased. In the distal caput and proximal corpus, large heavily stained droplets associated with spermatozoa were seen in the lumen. The staining intensity of these droplets also decreased from corpus to cauda. The clear cells of the distal corpus and cauda did not stain with the antibody to MEP 9. Sequential extraction of caudal spermatozoa showed that some MEP 9 was extractable under low-salt conditions, whereas extraction with 0.1% Triton X-100 was required to remove all MEP 9, indicating it was firmly associated with spermatozoa. The antibody to MEP 9 cross-reacted with a 25-kDa protein present in rat caudal fluid. MEP 10 was localized within the cytoplasm of the principal cells, the stereocilia, and the lumen of the epididymis at the junction of the distal caput and corpus. In the distal corpus, a large number of clear cells were stained, but very few of these cells stained in the cauda. MEP 10 dissociated completely from caudal spermatozoa under low-salt conditions, indicating that it was not firmly bound to spermatozoa. The antiserum to MEP 10 cross-reacted with proteins present in rat and guinea pig caudal fluid. The related rat protein migrated at approximately 20 kDa. Amino acid sequence analysis of MEP 10 revealed an 86% sequence similarity with rat proteins B and C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tn916 insertion mutagenesis in Escherichia coli and Haemophilus influenzae type b following conjugative transfer.

Transposon Tn916 was shown to be capable of direct conjugative transfer in broth and membrane matings between strains of Escherichia coli K12 and between E. coli K12 and Haemophilus influenzae type b. Only Tn916 was transferred, but Tn916 donor ability was not itself inheritable by the recipients and seemed to be associated with the presence of Tn916 on a non-conjugative pBR322-derived vector in the original donor strain. Transfer of Tn916 by conjugation was found to be an efficient method for producing insertion mutations in the chromosome of recipient cells. Although such insertions were unstable when the cells were grown under non-selective conditions, it was possible to show that over 40% of the isolated Tn916 insertions in the chromosome of E. coli K12 were in gene(s) concerned with histidine biosynthesis, implying that there is a partial hot-spot for Tn916 insertion on the E. coli K12 chromosome. When a strain of H. influenzae type b was used as a recipient, out of approximately 1500 transconjugants tested, two mutants were isolated with insertions in genes controlling the expression of iron-regulated transferrin-binding proteins. These mutants constitutively produced major 76 kDa and minor 90 kDa proteins which bound transferrin, even when grown under iron-sufficient conditions. Tn916 insertion mutagenesis, following transfer by conjugation, is a convenient method for isolating mutations in genes concerned with iron acquisition by this important human pathogen.     

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.     

Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants

Mutation at either of two genetic loci (Eu2 or Eu3) in soybean (Glycine max [L.] Merr.) results in a pleiotropic elimination of the activity of both major urease isozymes. Surprisingly, the phenotype of a phylloplane bacterium, Methylobacterium mesophilicum, living on the leaves of eu2/eu2 or eu3-e1/eu3-e1 mutants is also affected by these plant mutations. The bacteria isolated from leaves of these soybean mutants have transient urease- and hydrogenase-deficient phenotypes that can be corrected by the addition of nickel to free-living cultures. The same bacterium growing on wild-type soybeans or on urease mutants eu1-sun/eu1-sun or eu4/eu4, each deficient in only one urease isozyme, are urease-positive. These results suggest that the bacterium living on the eu2/eu2 or eu3-e1/eu3-e1 mutant is unable to produce an active urease or hydrogenase because it is effectively starved for nickel. We infer that mutations at Eu2 or Eu3 result in defects in nickel metabolism but not in Ni²⁺ uptake or transport, because eu2/eu2 and eu3-e1/eu3-e1 mutants exhibit normal uptake of ⁶³NiCl₂. Moreover, wild-type plants grafted on mutant rootstocks produce seeds with fully active urease, indicating unimpeded transport of nickel through mutant roots and stems.     

Calcium in bacteria: a solution to which problem?

Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.