The Fe-only nitrogenase from Rhodobacter capsulatus: identification of the cofactor, an unusual, high-nuclearity iron-sulfur cluster, by Fe K-edge EXAFS and 57Fe Mössbauer spectroscopy.

Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by 57Fe Mössbauer spectroscopy and by Fe and Zn EXAFS as well as XANES spectroscopy. The analyses were performed on the basis of data known for the FeMo cofactor and the P cluster of Mo nitrogenases. The prominent Fourier transform peaks of the Fe K-edge spectrum are assigned to Fe-S and Fe-Fe interactions at distances of 2.29 A and 2.63 A, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 A, which is an unprecedented feature of the trigonal prismatic arrangement of iron atoms found in the FeMo cofactor of nitrogenase MoFe protein crystal structures. Additional Fe...Fe interactions at 2.92 A and 4.05 A clearly indicate that the principal geometry of the P cluster is also conserved. Mössbauer spectra of 57Fe-enriched FeFe protein preparations were recorded at 77 K (20 mT) and 4.2 K (20 mT, 6.2 T), whereby the 4.2 K high-field spectrum clearly demonstrates that the cofactor of the Fe-only nitrogenase (FeFe cofactor) is diamagnetic in the dithionite-reduced ("as isolated") state. The evaluation of the 77 K spectrum is in agreement with the assumption that this cofactor contains eight Fe atoms. In the literature, several genetic and biochemical lines of evidence are presented pointing to a significant structural similarity of the FeFe, the FeMo and and the FeV cofactors. The data reported here provide the first spectroscopic evidence for a structural homology of the FeFe cofactor to the heterometal-containing cofactors, thus substantiating that the FeFe cofactor is the largest iron-sulfur cluster so far found in nature.

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

Expression of the blistered/DSRF gene is controlled by different morphogens during Drosophila trachea and wing development.

The Drosophila serum response factor (DSRF) is expressed in the precursors of the terminal tracheal cells and in the future intervein territories of the third instar wing imaginal disc. Dissection of the DSRF regulatory region reveals that a single enhancer element, which is under the control of the fibroblast growth factor (FGF)-receptor signalling pathway, is sufficient to induce DSRF expression in the terminal tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is active in the central intervein sector and is induced by the Hedgehog signalling pathway. The other element is under the control of Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector. Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and the localized expression of a gene required for the final differentiation of this territory.

Biochemical and biophysical characterization of refolded Drosophila DPP, a homolog of bone morphogenetic proteins 2 and 4.

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15 degreesC). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coli-expressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor-beta superfamily members, shown to act as intramolecular chaperones in vivo.

The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered.

The adult Drosophila wing is formed by an epithelial sheet, which differentiates into two non-neural tissues, vein or intervein. A large number of genes, many of them encoding components of an EGF-receptor signaling pathway, have previously been shown to be required for differentiation of vein tissue. Much less is known about the molecular control of intervein differentiation. Here we report that the Drosophila homolog of the mammalian Serum Response Factor gene (DSRF), which encodes a MADS-box containing transcriptional regulator, is expressed in the future intervein tissue of wing imaginal discs. In adult flies carrying only one functional copy of the DSRF gene, additional vein tissue develops in the wing, indicating that DSRF is required to spatially restrict the formation of veins. In mitotic clones lacking DSRF, intervein tissue fails to differentiate and becomes vein-like in appearance. Genetic and molecular evidence demonstrates that DSRF is encoded by the blistered locus, which produces ectopic veins and blistered wings when mutant. Our results show that DSRF plays a dual role during wing differentiation. It acts in a dosage-dependent [correction of dosage-dependant] manner to suppress the formation of wing veins and is required cell-autonomously to promote the development of intervein cells. We propose that DSRF acts at a key step between regulatory genes that define the early positional values in the developing wing disc and the subsequent localized expression of intervein-specific structural genes.

The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system.

We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

Sequence-independent detection of gene family homologs: identification of a transcript encoding a molluscan serine protease homologous to the pancreatic enzymes of vertebrates.

Autoradiography of 32P-labeled cDNA, fractionated at high resolution by electrophoresis through thin (0.8-1.5 mm) vertical alkaline agarose gels, provides a sequence-independent screening procedure for gene family homologs. A screen of tissues of a marine mollusc revealed a prominent intestine-specific cDNA encoding a pancreatic serine protease homolog, which was not detectable as a discrete poly(A)+ RNA species on formaldehyde agarose gels. Discrete cDNA products are authentic, non-truncated transcripts of tissue-specific mRNA. A band-sharpening effect is imparted to cDNA products due to (a) substitution of a uniform length 5'-oligo(dT) terminus for heterogeneous 3'-poly(A) termini and (b) the inherent superior resolution of alkaline-denatured DNA.

The Drosophila SRF homolog is expressed in a subset of tracheal cells and maps within a genomic region required for tracheal development.

The Drosophila homolog of the vertebrate serum response factor (SRF) was isolated by low stringency hybridization. Nucleotide sequence analysis revealed that the Drosophila SRF homolog (DSRF) codes for a protein that displays 93% sequence identity with human SRF in the MADS domain, the region required for DNA binding, dimerization and interaction with accessory factors. The DSRF gene is expressed during several phases of embryonic development. In the egg, both the RNA and the protein are maternal in origin and slowly decrease in amount during gastrulation. After germ band retraction, high levels of zygotic expression are observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. Many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues. The DSRF gene was mapped to position 60C on the second chromosome, and overlapping deficiencies which remove the gene were identified. Analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. Although the initial migration of tracheal cells was not affected in those deficient embryos, many tracheal cells appeared not to maintain their correct position and continued to migrate. Thus, the DSRF gene might play a role in the proper formation and maintenance of the trachea.

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air. Finally, AFM images are presented of single-stranded phi X-174 virion DNA with the gene 32 single-stranded binding protein. A summary of the current state of the field and of the methods for preparing and imaging DNA in the AFM is also presented.

Molluscan chymotrypsin-like protease: structure, localization, and substrate specificity.

A messenger RNA encoding a chymotrypsin-like preproprotease is expressed abundantly and specifically in the distal quarter of the intestine of the mollusc Haliotis rufescens (red abalone). Consistent with this finding, a chymotrypsin-like activity was detected at highest concentration in the lumen of this segment of the intestine. Because the complexity of total protein in the distal intestinal fluid was low and the chymotrypsin-like protease was highly expressed, purification of the enzyme to near homogeneity was achieved by a single passage over an anion-exchange resin. The primary specificity of the protease, predicted from homology of the key amino acid residues (Ser189, Gly216, and Ser226) in the substrate binding site with similar residues lining the S1 subsites of other chymotrypsin-like enzymes, was confirmed by hydrolysis of a family of tetrapeptide substrates with different P1 amino acids. The optimal P1 residues include those with bulky, gamma-branched side chains (phenylalanine and leucine) similar to the side chain of the asparagine residue at P1 of the activation peptide of the proenzyme. Thus, unlike zymogens of the pancreatic serine proteases, which are activated by the common tryptic mechanism, the zymogen of the molluscan enzyme appears to be activated by an autocatalytic mechanism, i.e., by cleavage with active chymotrypsin-like protease. Additional unique properties of the enzyme predicted from the primary sequence include an unpaired cysteine, with the potential for internal thiol-disulfide isomerization between two different conformers of the protease.

Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans.

RNA isolated by conventional guanidinium isothiocyanate methods from tissues of a mollusc (red abalone: Haliotis rufescens) is largely degraded and discolored by contaminants. These contaminants are associated with inhibition of reverse transcriptase, prevent accurate spectrophotometric determination of RNA concentration, and impart undesirable viscosity to the preparations. A cold two-step method of RNA isolation was devised which provides high yields of full-length RNA templates from these tissues and eliminates the discolored contaminant. Immediately following homogenization of tissues at ca. 5 degrees C, which proved crucial for the recovery of high-molecular weight species, the RNA is isolated from the bulk of the RNase by a single acid-phenol-chloroform extraction at 0 degrees C. The inhibitor of reverse transcriptase, suspected to be a proteoglycan (or a similar high-molecular-weight polyanion) component of the intestinal mucus, is eliminated only by a second purification step employing ultracentrifugation through a dense cushion of CsCl. This cold two-step method should prove useful for providing full-length RNA templates relatively free of polysaccharide, a common contaminant of RNA preparations, from both plant and animal tissues.

Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments.

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).