Cells are added to the archenteron during and following secondary invagination in the sea urchin Lytechinus variegatus.

In the present investigation, nuclei of endodermal cells, primary and secondary mesenchyme cells (PMCs and SMCs), and small micromere descendants (SMDs) of the sea urchin Lytechinus variegatus were counted and mapped at five developmental stages, ranging from primary invagination to pluteus larva. The archenteron and its derivatives were measured three dimensionally with STERECON analytical software. For the first time SMC production is included in the kinetic analysis of archenteron formation. While the archenteron lumen doubled in length during secondary invagination, the number of archenteron cells increased by at least 38% (over 50% when SMCs that emigrated from the tip of the archenteron were included). The volume of the archenteron epithelial wall plus the volume of 17 new SMCs increased by 40% over the equivalent volumes at the end of primary invagination. Because secondary invagination involves the addition of archenteron cells and an increase in volume of the archenteron epithelium, we conclude that secondary invagination is not accomplished simply by the rearrangement and reshaping of the primary archenteron cells. Both archenteron cell number and wall volume continued to increase at the same rates from the end of secondary invagination until the 27-h prism stage, although the lumen lengthened more slowly. SMCs were also produced at a constant rate from primary invagination until the prism stage. Because the production of both endodermal and mesodermal cells continues until the late prism stage, we conclude that gastrulation (defined as the establishment of the germ layers) also extends into the late prism stage.

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

Photosynthetic Gene Expression in Meristems and during Initial Leaf Development in a C4 Dicotyledonous Plant.

Immunolocalization and fluorescent in situ hybridization were used with confocal microscopy to examine patterns of photosynthetic gene expression during initial stages of leaf development in the C4 dicot Amaranthus hypochondriacus. mRNAs encoding the large and small subunit of ribulose-1,5-bisphosphate carboxylase were present in the apical dome and in all cells of the leaf primordia. In contrast, these polypeptides were detected only in cells of the ground meristem, with no accumulation detected in the apical dome or in other leaf primordia cells. The ribulose-1,5-bisphosphate carboxylase transcripts showed very little cell-type specificity as leaf structures began to differentiate, whereas their polypeptides accumulated primarily in bundle-sheath precursor cells. Phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase mRNAs were abundant in meristems and leaf primordia, although their corresponding polypeptides did not accumulate in leaves until the leaf vascular system began to differentiate. These polypeptides were mostly restricted to premesophyll cells from their earliest detection, whereas their transcripts remained present in nearly all leaf cells. These findings indicate that individual C4 genes are independently regulated as they become initially localized to their appropriate cell types. Furthermore, posttranscriptional regulation plays a major role in determining early patterns of C4 gene expression.

Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein.

While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.

One blastomere of the two-cell stage sea urchin embryo (Lytechinus variegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientation of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two methods were used to mark the blastomere: in the first, the lipophilic carbocyanine dye DiIC16 was microinjected directly into the blastomere after first cleavage was completed. In the second, caged (nonfluorescent) fluorescein-dextran was microinjected into the single-celled zygote and uncaged (made fluorescent) in one of the blastomeres at the two-cell stage using two-photon excitation microscopy (TPEM). This is the first use of TPEM for embryonic lineage tracing. In both methods the dye proved to be nontoxic and fluorescence was confined to lineally related cells. The results from both methods were similar and showed that the first cleavage furrow was variable in its orientation. Results were similar using animals obtained from different geographic locations. These results differ from those of McCain and McClay (Development, 1994, 120, 395-404), who reported that the median orientation was invariant in this species. The differences between the two studies are discussed. We conclude that first cleavage does not specify nor is it predictive of the bilateral axes in this species. The technique of TPEM is proffered as a powerful new tool that will enable the marking and tracing of embryonic cell lineages with less injury and more precision than current methods.

Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis.

Streptomyces glaucescens, a Gram-positive soil bacterium, produces the polyketide antibiotic tetracenomycin (Tcm) C. To study possible biochemical connections between the biosynthesis of bacterial fatty acids and polyketides, the abundant acyl carrier protein (ACP) detected throughout the growth of the tetracenomycin (Tcm) C-producing S. glaucescens was purified to homogeneity and found to behave like many other ACPs from bacteria and plants (apparent M(r) of 20,000 on gel filtration chromatography, apparent M(r) of 3400-4800 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and pI approximately 3.8). By using an oligodeoxynucleotide synthesized in accordance with the sequence of residues 25-36 of the ACP, the fabC gene encoding this protein was cloned, and expression of this gene in Escherichia coli yielded the ACP entirely as the active holoenzyme. Sequence analysis of 4.3 kilobases (kb) of DNA flanking fabC revealed the presence of three other genes oriented in the same transcriptional direction in the order fabD, fabH, fabC, and fabB. Each of the four genes is predicted to encode proteins with high sequence similarity to the following components of the E. coli fatty acid synthase (FAS): the FabD malonyl-coenzyme A:ACP acyltransferase (MAT), FabH 3-oxoacyl:ACP synthase III, AcpP ACP, and FabB 3-oxoacyl:ACP synthase I. Expression of the S. glaucescens fabD gene in E. coli produced active MAT able to catalyze in vitro the transfer of radioactive malonate from malonyl-coenzyme A to the E. coli AcpP and S. glaucescens FabC ACPs, as well as to the TcmM ACP component of the Tcm type II polyketide synthase [Shen, B., et al. (1992) J. Bacteriol 174, 3818-3821]. Expression of fabD also restored the high-temperature growth of the E. coli fabD89 mutant that bears a temperature-sensitive MAT. The latter finding and the close similarity between the organization of the S. glaucescens fabDHCB and E. coli FAS-encoding genes (fabH/fabD/fabG/acpP/fabF) suggest that the S. glaucescens genes encode FAS enzymes. Moreover, on the basis of its in vitro activity, it is possible that the S. glaucescens FabD MAT is responsible for charging the TcmM ACP with malonate in vivo, a key step in the synthesis of the deca(polyketide) precursor of Tcm C. This implies the existence of a functional connection between fatty acid and polyketide metabolism in this bacterium.

Overproduction of the acyl carrier protein component of a type II polyketide synthase stimulates production of tetracenomycin biosynthetic intermediates in Streptomyces glaucescens.

The development of microorganisms with improved antibiotic production is an important goal in the commercialization of new pharmaceuticals or in lowering the cost of established drugs. We report a way to achieve this for biosynthetic intermediates of an antibiotic made by the polyketide pathway whose earliest steps involve a Type II multienzyme complex. Introduction of the tcmKLM beta-ketoacyl: ACP synthase and acyl carrier protein (ACP) genes or just the tcmM ACP gene into the tetracenomycin (Tcm) C-producing Streptomyces glaucescens wild-type strain, or its tcmN or tcmO blocked mutants, on high copy vectors under the control of strong promoters caused a 2 to 30-fold overproduction of Tcm D3 and some other biosynthetic intermediates (or shunt products) and a 25 to 30% increase in Tcm C production relative to the control strains carrying the plasmid vector only. However, Tcm C production was not greater than that obtained with the vector-free wild-type strain. The unexpected effect of increased ACP on Tcm D3 production suggests that the level of this protein can influence either the activity or level of the three other components of the Tcm polyketide synthase.

The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase.

Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase.

Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group.

Preservation and visualization of the sea urchin embryo blastocoelic extracellular matrix.

Several methods were utilized to visualize the structure and orientation of the blastocoelic extracellular matrix (ECM) in Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Rapid freezing in liquid propane cooled to LN2 temperatures followed by freeze substitution was used to preserve the ECM without shrinkage due to dehydration. Scanning, transmission, and light microscopy were employed to elucidate the ECMs' structure. The blastocoelic ECM consisted of parallel fibrillar sheets that were interconnected by finer filaments and oriented along the animal-vegetal axis. The ECM completely filled the blastocoelic cavity as viewed by scanning electron microscopy. The basal lamina could be distinguished from the blastocoelic ECM as a thin coat on the plasma membrane of epithelial cells; the ECM was in contact with this coat. In contrast, the blastocoelic ECM attached directly to the plasma membrane of primary mesenchyme cells (PMC) which did not possess a basal lamina. The blastocoelic ECM was isolated as an intact "bag" and probed in a hydrated state with Con A and alcian blue. Confocal microscopy confirmed that the entire blastocoel was filled with a fibrillar ECM. These approaches offer advantages for future studies of the ECMs of sea urchin embryos and their roles in gastrulation.

Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase.

The genetic and biochemical basis of polyketide metabolism in microorganisms and its role in drug discovery and development.

The possibilities for the design of new drug screening and development strategies directed to a specific objective on the basis of genetic engineering of microorganisms is discussed from two points of view. Firstly, results of work on genetic hybrids of Streptomyces species for the production of new metabolites such as mederrhodin (1) and aloespanoarin II (4) are described. Secondly, the enhanced production of known metabolites such as tetracenomycin A2 (11) and tetracenomycin C (9) by recombinant Streptomyces species is considered. Mechanistic aspects of polyketide metabolism are included.

The genetic and biochemical basis of polyketide metabolism in microorganisms and its role in drug discovery and development.

The possibilities for the design of new drug screening and development strategies directed to a specific objective on the basis of genetic engineering of microorganisms is discussed from two points of view. Firstly, results of work on genetic hybrids of STREPTOMYCES species for the production of new metabolites such as mederrhodin (1) and aloespanoarin II (4) are described. Secondly, the enhanced production of known metabolites such as tetracenomycin A (2) (11) and tetracenomycin C (9) by recombinant STREPTOMYCES species is considered. Mechanistic aspects of polyketide metabolism are included.

The use of confocal microscopy and STERECON reconstructions in the analysis of sea urchin embryonic cell division.

A laser scanning confocal microscope has been used to investigate the development of the sea urchin embryo. The samples were fixed in Carnoy's solution at various developmental stages, stained for DNA with the Feulgen reaction, and optically sectioned with a BioRad MRC-500 confocal microscope. Computer-generated stereographic projection images and a three-dimensional contour tracing and reconstruction system were employed to investigate the cleavage pattern during the 6th cleavage division. Cell division is found to be asynchronous during the 6th cleavage, with macromere derivatives completing division first, followed by mesomeres, and finally by the outer quartet of micromeres (which begins division only after macromeres and mesomeres have completed their respective divisions). Sixth cleavage produces an embryo comprising 60 cells. Asynchronous division was also observed within individual tiers of blastomeres. Variations in the orientations of cell division axes within individual tiers of cells were also observed. The utility of computer-graphics reconstruction techniques for both quantitative and qualitative developmental analysis are discussed.

Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein.

The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.

A conservative amino acid substitution, arginine for lysine, abolishes export of a hybrid protein in Escherichia coli. Implications for the mechanism of protein secretion.

A hybrid protein that comprises the beta-lactamase signal peptide fused precisely to chicken muscle triosephosphate isomerase is not secreted into the periplasm of Escherichia coli. The protein can be secreted, however, if an arginine residue at position 3 of the isomerase is replaced by either a serine or a proline residue. In contrast, replacement of a neighboring lysine residue has no effect on secretion of the protein. Furthermore, if the arginine is removed from position 3 to generate a secreted protein, but is then reintroduced in place of the neighboring lysine, the blockade to secretion is re-established. The singular effect of the arginine residue on secretion does not result from the role this residue plays in the formation or stabilization of the native isomerase structure: mutational alterations remote from the N terminus of the isomerase that prevent the proper folding of the protein do not relieve the block to secretion. The finding that an arginine residue prevents secretion while a lysine residue does not, suggests that basic residues near the mature N terminus of a secreted protein must be deprotonated if orderly export is to occur. This implies that the signal peptide along with the N-terminal portion of the mature protein partitions directly into the lipid bilayer in the course of the secretory process.

Secretion of beta-lactamase into the periplasm of Escherichia coli: evidence for a distinct release step associated with a conformational change.

The secretion of beta-lactamase (EC 3.5.2.6) into the periplasm of Escherichia coli has been followed by pulse-chase labeling at 15 degrees C. Though the periplasmic fraction contains only the mature form of the enzyme, the spheroplast fraction contains the completed precursor and a hitherto undocumented processed form. When whole spheroplasts are treated with trypsin, the processed form in this fraction is completely digested. This is in contrast to the native mature enzyme localized in the periplasm, which is trypsin resistant. The beta-lactamase is evidently processed after translocation to a trypsin-sensitive form that is transiently bound to the periplasmic face of the inner membrane. The release of this processed form into the periplasm occurs concomitantly with a conformational change that results in the soluble, catalytically active, trypsin-resistant structure.

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis.

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.