U1A inhibits cleavage at the immunoglobulin M heavy-chain secretory poly(A) site by binding between the two downstream GU-rich regions

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.     

Predetermination of cytokinesis and transition from holoblastic to partial superficial cleavage in early embryonic development of Tetrodontophora bielanensis (Collembola)

In the T. bielanensis embryo, only karyokinesis occurs during the first cleavage division, and a two-nuclear syncytial embryo forms. Then, two cytoplasmic concentrations in the form of elongated rolls perpendicular to each other develop below the periplasm at the animal pole of the egg. The second cleavage division is also associated with karyokineses only. After the embryo reaches the four-nuclear stage, cytokinesis occur at its animal pole, and two cleavage furrows perpendicular to each other develop in the periplasm above the cytoplasmic concentrations. The cell membranes forming within the furrows do not invade the cytoplasmic concentrations, but their growing tips push them into the egg interior, where they merge and form the central cytoplasmic concentration. The developing cell membranes do not invade the central cytoplasm; they band and grow above its surface. Four pyramidal blastomeres form as a result of this. The eight-blastomere embryo forms through both karyokinesis and cytokinesis, but the growing cell membranes now band below the previous ones and cut off anucleate parts of the mother blastomeres, which fuse with the central cytoplasm. Thus, during this phase of development the transition from holoblastic to partial superficial cleavage is initiated. Morphological analysis suggests that the formation of the first two cytokinesis is predetermined by and depends on factors connected with the animal pole periplasm. It also suggests that the central cytoplasm constitutes the morphological field, inducing the transition from holoblastic to partial superficial cleavage.     

Membrane-bound beta-lactamase forms in Escherichia coli

Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site. These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane. The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth. We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane. Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se. If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane. If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm. If only the first signal contains a cleavage site, a longer fusion protein is transported and released. The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

Monitoring the Disulfide Bond Formation of a Cysteine-Rich Repeat Protein from Helicobacter pylori in the Periplasm of Escherichia coli

Helicobacter pylori infection increases the risk of cardiovascular diseases besides leading to duodenal and gastric peptic ulcerations. H. pylori cysteine-rich protein B (HcpB) is a disulfide-rich repeat protein that belongs to the family of Sel1-like repeat proteins. HcpB contains four pairs of anti-parallel alpha helices that fold into four repeats with disulfide bonds bridging the helices of each repeat. Recent in vitro oxidative refolding of HcpB identified that the formation and folding of the disulfide bond in the N-terminal repeat are the rate limiting step. Here we attempted to understand the disulfide formation of HcpB in the periplasm of Escherichia coli. The protein was expressed in wild type (possessed enzymes DsbA, B, C, and D) and knock out (Dsb enzymes deleted one at a time) E. coli strains. The soluble part of the periplasm when analyzed by SDS-PAGE and Western Blot showed that the wild type and DsbC/D knock out strains contained native oxidized HcpB while the protein was absent in the DsbA/B knock out strains. Hence the recombinant expression of HcpB in E. coli requires DsbA and DsbB for disulfide bond formation and it is independent of DsbC and DsbD. Prolonged cell growth resulted in the proteolytic degradation of the N-terminal repeat of HcpB. The delayed folding of the N-terminal repeat observed during in vitro oxidative refolding could be the reason for the enhanced susceptibility to proteolytic cleavage in the periplasm. In summary, a good correlation between in vivo and in vitro disulfide bond formation of HcpB is observed.     

Naturally occurring human parainfluenza type 3 viruses exhibit divergence in amino acid sequence of their fusion protein neutralization epitopes and cleavage sites.

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.     

4株鹅源新城疫病毒融合蛋白基因的克隆及序列分析

测定了4株鹅源新城疫病毒(NDV)融合蛋白(F)基因5’端1700核苷酸片段的序列,并由此推导了F蛋白氨基酸序列,并对鹅源NDV的基因型分类地位进行探讨。结果表明,4株病毒F基因的同源性大于97%,与DNV标准强毒株F48E8 F基因的同源性为860%～868%,F基因转录起始序列及起始密码子位置与已知NDV完全相同;F蛋白具有和已知NDV相似的各种功能区,F蛋白前体F0裂解位点附近的氨基酸序列为112RRQKRF117,符合NDV强毒株的特征。对F基因第334～1682位核苷酸之间3种限制性内切酶HinfⅠ、BstoⅠ\,\%Rsa\%Ⅰ酶切图谱的分析表明,4株病毒的基因型与文献报道的I～Ⅷ型有明显差异。     

Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

The direction of cleavage waves and the regional variation in the duration of cleavage cycles on the dorsal side of the Xenopus laevis blastula

Summary The animal and the dorsal side of five embryos of Xenopus laevis were studied in detail from the 7th to the 13th cleavage by means of time-lapse cinematography. At each cleavage the regionally ordered sequence of blastomere divisions is visible in the films as a cleavage wave, propagating about three times slower in the dorsal than in the animal view. In the dorsal view the waves run in an animal-vegetal direction, initially with a left-to-right deviation and in later cleavages converging on the region of the future blastopore. The lengthening of cleavage cycles begins at cycle 8 on the dorsal side, just above the future blastopore. From cycle 9 to 11 nearly equal lengthening occurs in each cycle at all animal-vegetal levels. In general, cycles lengthen a little more in median than in lateral sectors and a little more in right than in left sectors. Cycle 12 is longest in the sector above the future blastopore and shortest in the animal region. The results show that the initial pattern of a regionally ordered sequence of cleavage cycles of equal duration changes into a pattern of cycles of different durations as a result of gradual cycle lengthening, starting in the region just above the future blastopore and spreading in animal direction. The results are compared with data on the cleavage cycles of isolated blastomeres, and the possible relation with the induction of the mesoendoderm occurring during the stages studied is discussed.     

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

Periodic conformational changes in rRNA: monitoring the dynamics of translating ribosomes

In protein synthesis, a tRNA transits the ribosome via consecutive binding to the A (acceptor), P (peptidyl), and E (exit) site; these tRNA movements are catalyzed by elongation factor G (EF-G) and GTP. Site-specific Pb2+ cleavage was applied to trace tertiary alterations in tRNA and all rRNAs on pre- and posttranslocational ribosomes. The cleavage pattern of deacylated tRNA and AcPhe-tRNA changed individually upon binding to the ribosome; however, these different conformations were unaffected by translocation. On the other hand, translocation affects 23S rRNA structure. Significantly, the Pb2+ cleavage pattern near the peptidyl transferase center was different before and after translocation. This structural rearrangement emerged periodically during elongation, thus providing evidence for a dynamic and mobile role of 23S rRNA in translocation.     

Shh-dependent formation of the ZLI is opposed by signals from the dorsal diencephalon

The zona limitans intrathalamica (ZLI) is located at the border between the prospective ventral thalamus and dorsal thalamus, and functions as a diencephalic signaling center. Little is known about the mechanism controlling ZLI formation. Using a combination of fate-mapping studies and in vitro assays, I show that the differentiation of the ZLI from progenitor cells in the alar plate is initiated by a Shh-dependent signal from the basal plate. The subsequent dorsal progression of ZLI differentiation requires ongoing Shh signaling, and is constrained by inhibitory factors derived from the dorsal diencephalon. These studies demonstrate that self-organizing signals from the basal plate regulate the formation of a potential patterning center in the ZLI in an orthogonal orientation in the alar plate, and thus create the potential for coordinated thalamic patterning in two dimensions.     

A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus. One of the dorsal group genes, dorsal (dl), encodes the putative morphogen. Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg. At the dorsal side of the egg, the protein remains in the cytoplasm. Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg. A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.     

Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain

The division of the mammalian forebrain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic proteins (BMPs) have been implicated in dorsal midline development, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline development in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signaling plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression.     

Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells.

The Escherichia coli maltose-binding protein (MBP) R2 signal peptide is a truncated version of the wild-type structure that still facilitates very efficient export of MBP to the periplasm. Among single amino acid substitutions in the R2 signal peptide resulting in an export-defective precursor MBP (pMBP) were two that replaced residues in the consensus Ala-X-Ala sequence (residues -3 to -1) that immediately precedes the cleavage site. It was suggested that the functional hydrophobic core and signal peptidase recognition sequence of this signal peptide substantially overlap and that these two alterations affect both pMBP translocation and processing. In this study, the export of pMBP by the mutants, designated CC15 and CC17, with these two alterations was investigated further. The pMBP of mutant CC17 has an Arg substituted for Leu at the -2 position. It was found that CC17 cells exported only a very small amount of MBP, but that which was exported appeared to be correctly processed. This result was consistent with other studies that have concluded that virtually any amino acid can occupy the -2 position. For mutant CC15, which exhibits a fully Mal+ phenotype, an Asp is substituted for the Ala at the -3 position. CC15 cells were found to export large quantities of unprocessed, soluble pMBP to the periplasm, although such export was achieved in a relatively slow, posttranslational manner. This result was also consistent with other studies that suggested that charged residues are normally excluded from the -3 position of the cleavage site. Using in vitro oligonucleotide-directed mutagenesis, we constructed a new signal sequence mutant in which Asp was substituted for Arg at the -3 position of an otherwise wild-type MBP signal peptide. This alteration had no apparent effect on pMBP translocation across the cytoplasmic membrane, but processing by signal peptidase was inhibited. This pMBP species with its full-length hydrophobic core remained anchored to the membrane, where it could still participate in maltose uptake. The implications of these results for models of protein export are discussed.     

Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta

BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.     

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.     

DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.     

Ribonuclease A as a substrate of the protease from human immunodeficiency virus-1

Bovine pancreatic ribonuclease A (RNase) contains two bonds, Met29-Met30 and Tyr92-Pro93 which are representative of sites in the human immunodeficiency virus-1 (HIV-1) gag polyprotein precursors that are cleaved by the HIV-1 protease during viral maturation. Nevertheless, neither native nor performic acid-oxidized RNase is a substrate for the protease. However, RNase derivatives obtained by reduction and S-alkylation with iodoacetate or iodoacetamide undergo cleavage by the HIV-1 protease at a single site, Ala109-alkyl-Cys110, that is distinct from either of the two predicted bonds mentioned above. The neutral carboxyamido-methylcysteinyl derivative is cleaved 8 times faster than that containing the negatively charged carboxy-methyl substituent at P1'. Succinylation of these S-alkylated RNase derivatives creates a second site of cleavage by the protease between succinyl-Lys7 and Phe8. Thus, the pattern of cleavage of denatured RNase by the HIV-1 protease can be manipulated by chemical derivatization of the substrate, and the new sites of hydrolysis revealed by these studies add to our understanding of the specificity of this important enzyme.