The 1.5-nm projection structure of HeLa cell prosome-MCP (proteasome) provided by two-dimensional crystals.

We grew two-dimensional crystals of HeLa cell prosomes, also called multicatalytic proteinases (MCP) and proteasomes, for a structure determination by electron microscopy. The molecules were arranged in side views in these crystals. The crystals have p21 plane group symmetry with one particle per unit cell. This symmetry confirms previously published evidence indicating that eukaryotic prosome-MCPs are dimers of two identical halves. Structure factors from six crystals each comprising more than 1000 unit cells were combined to generate a 1.5-nm projection map. We discovered that while the general cylindrical shape of HeLa prosome-MCPs resembles the shape of the archaebacterial Thermoplasma acidophilum proteasomes, the internal structure differs significantly. We propose that because of different subunit composition, the architecture of HeLa prosome-MCPs differs from the basic architecture of related particles previously reported.

Isolation and characterization of the rat gene encoding glutamate dehydrogenase.

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction-fragment-length polymorphism for several enzymes. Promoter activity of the 5'-flanking sequence is shown by transient transfection experiments. The 5'-flanking sequence contains a TTAAAA sequence at position -29, instead of a consensus TATA box and, like many other TATA-less promoters, is characterized by a very high G + C content. In addition, consensus sequences for the binding sites of the transcription factors Sp1 and Zif268 are present in the G + C-rich upstream region.

Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication.

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.

Structure and RNA content of the prosomes.

Duck erythroblasts prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of approximately 720,000 +/- 50,000, a radius of gyration of 64 +/- 2 A and a hydrodynamic radius of approximately 86 A were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 A, a height of 170 A and a diameter of 40 A for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14.

The genes encoding the large (cfxL) and small (cfxS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisC/O) from Xanthobacter flavus H4-14 were identified and characterized. The RuBisC/O genes are separated by 11 bp and cotranscribed in Escherichia coli from the lac promoter in the order cfxLS. Primer extension and R-loop experiments with RNA isolated from autotrophically grown X. flavus H4-14 showed that transcription of cfxL and cfxS initiated 22 bp upstream from cfxL and resulted in a mRNA of at least 2.3 kb. DNA sequence analysis identified the start of an open reading frame transcribed divergently from cfxL, and displaying significant similarities with genes belonging to the lysR family of transcriptional activators. Downstream from cfxS an additional open reading frame was identified with unknown function. Expression studies showed that the genes encoding fructosebisphosphatase (cfxF) and phosphoribulokinase (cfxP) are located downstream from cfxLS. The cfxF and cfxP genes are cotranscribed in the same direction as cfxLS in the order cfxFP.

Isolation and characterization of the rat glutamine synthetase-encoding gene.

From a rat genomic library in phage lambda Charon4A, a complete glutamine synthetase-encoding gene was isolated. The gene is 9.5-10 kb long, consists of seven exons, and codes for two mRNA species of 1375 nucleotides (nt) and 2787 nt, respectively. For both mRNAs, full-length cDNAs containing a short poly(A) tract were identified. The sequences of the entire mRNA and of the exon-intron transitions were determined. The smaller mRNA is identical to the 5' 1375 nt of the long mRNA and contains the entire protein-coding region. The position of the transcription start point was mapped. Within the first 118 bp of promoter sequence, a (T)ATAA-box, a CCAAT-box and an SP1-binding site were identified.

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha.

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.

HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex.

Employing an exonuclease III protection assay we detected a protein in crude HeLa nuclear extracts binding, with apparent sequence specificity, to molecular ends of adenovirus type 2 (Ad2) DNA. This protein, designated nuclear factor IV (NFIV), was purified to homogeneity and was shown to be a hetero-dimer of 72,000 and 84,000 Mr. Binding to terminal Ad2 sequences was strongly enhanced by the presence of either of the sequence-specific DNA-binding proteins nuclear factor I and nuclear factor III. These proteins appeared to function as blockades for translocation of NFIV on DNA, thus producing apparent sequence specificity. In the absence of such a blockade, NFIV moved freely, without energy input, on any double-stranded DNA forming a regular DNA-multimeric protein complex as shown by methidiumpropyl EDTA footprinting and electron microscopy. Binding is completely dependent upon the presence of molecular ends. Evidence was obtained for a two-step mechanism in which termini are recognized by NFIV and used as a starting point for subsequent translocation. The possible functions of the protein in adenovirus DNA replication and in cellular processes requiring DNA termini are discussed.

Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives.

Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the R1162 mob region in cis. The formation of single-stranded circular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT).

Characterization of a polymorphism in the 3' part of the chicken vitellogenin gene.

An allele giving rise to a polymorphism within the 3' part of the chicken vitellogenin gene was cloned, sequenced, and compared to the previously cloned allele. The polymorphism is formed by a perfect copy of 343 bp from intron 32 in tandem array with a perfect copy of 244 bp from intron 33; this 587-bp element is inserted in a head-to-tail arrangement in intron 33. We propose a mechanism in which an unequal crossing-over resulted in a vitellogenin gene with two exons 33, one of which was subsequently deleted. Thus, intron 33 was enlarged by the tandem repeats without affecting the protein-encoding sequence of the gene. At the boundaries of the repeated elements, two short direct repeats are found that resemble the recombination signals of immunoglobulin genes. They may have had a key role in the formation of the new allele.

Studies on the structures of the normal and abnormal goat thyroglobulin genes.

We have cloned the thyroglobulin (Tg) gene of normal goats and goitrous goats which have a Tg synthesis defect. At the 5'-end of the gene, we studied cosmid clones covering a region from 20 kilobases (kb) upstream from the Tg gene to 42 kb into it. Electron microscopy and restriction mapping show that this part of the gene contains 20 exons of 90-1190 bp, in total 4.9 kb of exonic information (56% of the mRNA) split by 19 introns of 150-9100 bp. The exons comprise 12% of the 5' sequences cloned. At the 3'-end, 55 kb were cloned, containing 10 kb of the gene which comprises only 3 exons of 550 bp in total. Sequence analysis of the 3'-end of the normal and abnormal Tg genes has revealed one transition mutation 3' to the reading frame in a stem-loop structure region of the last exon near the poly(A) addition site. Analysis of the promoter site and the first 5 exons has revealed only one difference between the normal and goitrous Tg genes: a Ser----Leu transition in exon 5. We also found an insertion in the fifth intron of the abnormal gene.

Turnover products of the apo very low density lipoprotein II messenger RNA from chicken liver.

The mature apo Very Low Density Lipoprotein II (apo VLDLII) mRNA appears in chicken liver within a few hours after estrogen administration. Apart from this mRNA species, shorter RNA molecules hybridizing to apo VLDLII sequences have been detected in rooster liver upon estrogen stimulation. These molecules are present in the non-polyadenylated fraction of the total cellular- and polysomal RNA. Northern blotting and electron microscopy of R-loops were employed to show that these shorter RNA molecules are truncated at their 3'-end. The 3'-termini were further characterized by nuclease S1 analyses, and are located predominantly in the 3' untranslated region of the mRNA. Using a secondary structure model (Shelness and Williams, J. Biol. Chem. 260, 8637-8646, 1985), we show that the 3' termini map mainly in unpaired regions of the structure.

Self-splicing of a group II intron in yeast mitochondria: dependence on 5' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.

Interlocked RNA circle formation by a self-splicing yeast mitochondrial group I intron.

RNA containing the aI3 group I intron of the yeast mitochondrial gene encoding cytochrome oxidase subunit I shows self-splicing in vitro. The excised intron, comprising 1514 nucleotides, is partially split into an upstream portion, containing the intronic reading frame, and a downstream portion, containing the typical group I conserved sequence elements. Full-length intron RNA and intron parts occur in linear and circular form. In the transesterification reactions leading to circle formation, only the guanosine nucleotide added during splicing is removed. Reincubation of isolated, complete circular intron RNA under self-splicing conditions leads to formation of free subintronic RNA circles. Under similar conditions, purified linear intron RNA gives rise to a number of circular and linear products, one of which consists of interlocked subintronic RNA circles. These observations suggest that the intron RNA possesses a dynamic structure in which subtle alterations in folding result in the formation of RNA products with different topology.

The hepatitis delta (delta) virus possesses a circular RNA.

Hepatitis delta (delta) virus (HDV), a satellite virus of the hepatitis B virus (HBV), causes a severe form of viral hepatitis in humans. Here we present evidence based on electron microscopy and electrophoretic behaviour that HDV contains a single stranded circular RNA molecule. This is the first animal virus identified with a circular RNA genome. Circular RNAs have only been found in plant viruses. We have obtained a partial complementary DNA clone representing approximately 25% of the total genome of HDV. Analysis of this cDNA revealed similarity to two plant viruses that may explain the origin of the virus.

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.

Genes coding for the elongation factor EF-1 alpha in Artemia.

The elongation factor EF-1 alpha is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There are only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over 10(4) base pairs. Local rearrangements give rise to a number of gene variants. Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1 alpha genes and one or two different Drosophila genes, respectively. Nucleotide sequencing revealed signals for synthesis and processing of EF-1 alpha transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro.

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.

Formation of lariats and circles in self-splicing of the precursor to the large ribosomal RNA of yeast mitochondria.

Self-splicing of the precursor to large ribosomal RNA of yeast mitochondria leads not only to circles but also to lariats, structures that have not been observed before as products of self-splicing. Lariats were studied by electron microscopy after hybridization with an RNA complementary to the 3' half of the precursor. This leads to differentiation in at least two classes of lariats that vary in the position of the branch point. In all lariats the tail carries the 3' end, which suggests that a 5' end is used for branch formation with an internal nucleotide. The circles are formed from excised introns. They lack only three nucleotides encoded by mitochondrial DNA along with the 5'-terminal G added in the course of self-splicing. The diverse number of self-splicing products arising in vitro testifies to the considerable reactivity of this intron. The formation of lariats in an RNA catalyzed reaction may have implications for views on the mechanism of splicing of nuclear pre-mRNAs.