Showing papers in "The EMBO Journal in 1986"

The relation between the divergence of sequence and structure in proteins.

Abstract: Homologous proteins have regions which retain the same general fold and regions where the folds differ. For pairs of distantly related proteins (residue identity approximately 20%), the regions with the same fold may comprise less than half of each molecule. The regions with the same general fold differ in structure by amounts that increase as the amino acid sequences diverge. The root mean square deviation in the positions of the main chain atoms, delta, is related to the fraction of mutated residues, H, by the expression: delta(A) = 0.40 e1.87H.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

Abstract: The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.

Abstract: To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.

Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.

Abstract: We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo. We constructed a defective recombinant retrovirus in which the Escherichia coli beta-galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M-MuLV). Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue. Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types. Following injection of the virus into mid-gestation mouse embryos, clones of lacZ-positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion. We identified the cell types comprising a series of lacZ-positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells. In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.

Mitochondrial targeting sequences may form amphiphilic helices.

Abstract: Twenty three mitochondrial targeting sequences have been analysed with regard to their potential for forming amphiphilic helices It is shown that most if not all of these sequences can be expected to form helices with high hydrophobic moments in a suitable environment In the few cases studied so far, the segments of maximal hydrophobic moment coincide closely with 'critical' regions defined by deletions and point mutations

The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology.

Abstract: The amino acid distribution in membrane spanning segments and connecting loops in bacterial inner membrane proteins was analysed. The basic residues Arg and Lys are four times less prevalent in periplasmic as compared to cytosolic connecting loops, whereas no comparable effect is observed for the acidic residues Asp and Glu. Also, Pro is shown to be tolerated to a much larger extent in membrane spanning segments with their N-terminus pointing towards the cytosol than in those with the opposite orientation. The significance of these findings with regard to the mechanism of biogenesis of bacterial inner membrane proteins is discussed.

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Abstract: Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.

Using known substructures in protein model building and crystallography

Abstract: Retinol binding protein can be constructed from a small number of large substructures taken from three unrelated proteins. The known structures are treated as a knowledge base from which one extracts information to be used in molecular modelling when lacking true atomic resolution. This includes the interpretation of electron density maps and modelling homologous proteins. Models can be built into maps more accurately and more quickly. This requires the use of a skeleton representation for the electron density which improves the determination of the initial chain tracing. Fragment-matching can be used to bridge gaps for inserted residues when modelling homologous proteins.

The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors.

Abstract: A chicken oviduct cDNA clone containing the complete open reading frame of the oestrogen receptor (ER) has been isolated and sequenced. The mol. wt of the predicted 589-amino acid protein is approximately 66 kd which is very close to that of the human ER. Comparison of the human and chicken amino acid sequences shows that 80% of their amino acids are identical. There are three highly conserved regions; the second and third of which probably represent the DNA- and hormone-binding domains of the receptor. The putative DNA-binding domain is characterised by its high cysteine and basic amino acid content, and the hormone-binding domain by its overall hydrophobicity. These two domains of homology are also present in the human glucocorticoid receptor (GR) and the product of the avian erythroblastosis virus (AEV) gene, v-erbA, indicating that c-erbA, the cellular counterpart of v-erbA, belongs to a multigene family of transcriptional regulatory proteins which bind steroid-related ligands. The first highly conserved ER region is not present in the truncated v-erbA gene, but shares some homology with the N-terminal end of the GR. The function of the v-erbA gene product is discussed in relation to its homology with the ER and GR sequences.

Human basic fibroblast growth factor: nucleotide sequence and genomic organization.

Abstract: Clones encoding the angiogenic endothelial cell mitogen, basic fibroblast growth factor (FGF), have been isolated from human cDNA libraries made from kidney, fetal heart, fetal liver, term placenta, and a breast carcinoma. Basic FGF cDNA clones are present in these libraries at very low levels when compared to the quantity of the growth factor in the tissues. This observation, combined with the fact that several of the clones represent unspliced transcripts, suggests that cytoplasmic basic FGF mRNA is unstable and that the protein is stored in tissues. The amino acid sequence of human basic FGF, deduced from the sequence of these cDNAs and from genomic clones, is 99% homologous to that of bovine basic FGF, implying a strong selection pressure for maintenance of function and structure. As with the bovine factor, human basic FGF does not appear to have a signal peptide sequence. Southern blot analysis of human genomic DNA and mapping of the cloned gene shows that there is only one basic FGF gene. All of the basic, heparin-binding endothelial cell mitogens of similar amino acid composition that have been described must therefore be products of this single gene.

The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA.

Abstract: Glutathione peroxidase (GSHPx) is an important selenium-containing enzyme which protects cells from peroxide damage and also has a role in leukotriene formation. We report the identification of a genomic recombinant as encoding the entire mouse GSHPx gene. Surprisingly, the selenocysteine in the active site of the enzyme is encoded by TGA: this has been confirmed by primer extension/dideoxy sequencing experiments using reticulocyte mRNA. The same site of transcription initiation is used in three tissues in which the GSHPx mRNA is expressed at high levels (erythroblast, liver and kidney). Like some other regulated 'house-keeping' genes, the GSHPx gene has Sp1 binding site consensus sequences but no 'ATA' and 'CAAT' consensus sequences upstream of the transcription initiation site. Moreover, there is a cluster of two Sp1 binding site consensus sequences and two SV40 core enhancer sequences in the 3' region of the gene, close to the previously mapped position of a DNase I-hypersensitive site found only in tissues expressing the GSHPx mRNA at high levels.

The integrase family of site-specific recombinases: regional similarities and global diversity.

Abstract: A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1 This group of proteins exhibits an unexpectedly large diversity of sequences Despite this diversity, all of the recombinases can be aligned in their C-terminal halves A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein This family of recombinases does not appear to be related to any other site-specific recombinases Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining

Pigment-protein interactions in the photosynthetic reaction centre from Rhodopseudomonas viridis.

Abstract: An X-ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment-binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (`special pair') and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light-driven electron transfer. The primary electron acceptor, a menaquinone (QA), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone QB (o-phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.

Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor.

Abstract: Site-directed mutagenesis was used to prepare a series of human oestrogen receptor (hER) deletion mutants. The ability of these mutant receptors to bind oestradiol, either after being transiently expressed in HeLa cells or produced synthetically in vitro using T7 polymerase coupled with a rabbit reticulocyte lysate translation system, was analysed. The results indicate that a region which is highly conserved (94% amino acid identity) between the human and chicken ERs (region E) contains all of the sequence necessary to bind oestradiol with high affinity. When tight nuclear association of the oestradiol-receptor complex was investigated using the oestradiol-binding mutants of the same series, two regions of the hER sequence were found to be important. One of these regions is completely conserved (100% amino acid identity) between the human and chicken ERs (region C). This region is rich in cysteine and basic amino acids and contains motifs similar to those which have been proposed to be important for DNA binding in other eukaryotic transcriptional regulatory proteins. The other region (region D), which is comparatively poorly conserved (38% amino acid identity), is located between the putative DNA-binding domain (region C) and the oestradiol-binding domain (region E).

Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines.

Abstract: Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3-kb RNA designated IFN-beta 2 and distinct from the 0.9-kb IFN-beta 1 mRNA. In vitro transcription--translation mapping of the full-length IFN-beta 2 cDNA sequence, shows that it encodes a 23.7-kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN-beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN-beta 2. Specific immunoassays show that IFN-beta 2 secreted by DNA-transformed rodent cells is a processed 21-kd protein, whose activity is cross-neutralized by antibodies to human IFN-beta 1 but not to IFN-alpha or gamma. The immunoassay also demonstrates the induction of IFN-beta 2 secretion by fibroblasts in response to growth-regulatory cytokines, such as interleukin-1 and tumor necrosis factor. The function of this IFN-beta 2 as an autoregulatory inhibitor of cell growth is discussed.

Induction of interleukin 2 receptor gene expression by p40x encoded by human T-cell leukemia virus type 1.

Abstract: Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia (ATL). A viral product, p40x, encoded by the pX sequence of HTLV-1 is a trans-acting transcriptional activator of the long terminal repeat (LTR) and has been suspected of involvement in leukemogenesis, activating the cellular genes. The cellular interleukin-2 (IL-2) and its receptor (IL-2R), the latter of which is expressed on ATL leukemic cells, were shown to be transiently induced by transfection of plasmid pMTPX expressing pX in two T-cell lines, Jurkat and HSB-2, but not in other human T- or B-cell lines. The cell type specificity of IL-2R induction by pX expression was the same as that by phytohaemagglutinin/phorbol ester activation, indicating the requirement for some specific cellular factors or a certain state of cellular differentiation. Induction of IL-2 and IL-2R at mRNA level was also demonstrated in transfected cells. Transfections with mutants of pMTPX in which the open reading frames for p40x, p27x-III and p21x-III were inactivated indicated that p40x alone was sufficient for induction of the IL-2R in inducible cells. This induction of the IL-2R by p40x of HTLV-1 may contribute to preferential proliferation of HTLV-1 infected cells at an early stage of ATL development and eventually increase the number of putative target cells for malignant transformation.

Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.

Abstract: Biosynthesis of HLA class I antigens has been studied in a variant B-LCLxT-LCL hybrid, 174XCEM.T2. This cell line encodes HLA-A2 and -B5, but expresses only small amounts of A2 antigen and undetectable B5 antigen at the cell surface due to a mutation inactivating a trans-acting regulatory gene encoded within the class II region of the human major histocompatibility complex. Northern blot analysis with HLA-A- and HLA-B-specific probes shows that 174XCEM.T2 synthesizes quantities of A and B locus mRNA comparable with its class I antigen-positive parent cell line. Immune precipitation studies indicate that 174XCEM.T2 synthesizes normal HLA heavy chains and beta 2-microglobulin which fail to form dimers. The heavy chains are N-glycosylated normally, but processing of the glycan to the complex form does not occur. In addition, free heavy chains in this cell line are not phosphorylated. Thus, the majority of class I heavy chains in 174XCEM.T2 do not combine with beta 2-microglobulin, and are not processed or transported to the cell surface. As both subunits are synthesized in normal amounts, we propose that an additional molecule absent from 174XCEM.T2 and encoded by an HLA-linked gene is necessary for efficient assembly of class I antigen subunits.

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells

Abstract: Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.

Different Human Cervical Carcinoma Cell Lines Show Similar Transcription Patterns of Human Papillomavirus Type 18 Early Genes

Abstract: Transcription of human papillomavirus type 18 (HPV18) DNA in the human cervical carcinoma cell lines HeLa, C4-1 and SW756 was studied by nucleotide sequence analysis of HPV18-positive cDNA clones isolated from a HeLa, C4-1 and SW756 cDNA library, respectively, and the cDNA sequences were used to predict the potential encoded proteins. The cDNA clones from all three cell lines were found to be derived from virus-cell fusion transcripts in which 3'-terminal host cell sequences (different for each cell line) were spliced to 5'-terminal exon sequences from the HPV18 E6-E7-E1 region. Three different types of cDNA clones can be distinguished according to the splicing patterns observed in the 5' terminal HPV18 sequences. They carry as potential protein-coding regions the HPV18 specific open reading frames E6 and E6* (generated by splicing and identical with E6 up to the E6* splice junction), E7 and E1 (only in HeLa). Translation of specific cellular genes from the chimeric viral-cellular transcripts seems to be unlikely. The mapping of the 5'-ends of the virus-cell fusion transcripts indicates that transcription is initiated at a viral promoter. The similar patterns of HPV18 transcription in the three different cervical carcinoma cell lines suggest a functional role of HPV18 early genes for the malignant phenotype of these cells.

A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers.

Abstract: Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino-terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha-helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.

Structure of the IgG-binding regions of streptococcal protein G.

Abstract: The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.

Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments.

Abstract: Fibronectin has been shown previously to promote complete cell adhesion in the absence of other serum components or de novo protein synthesis. Recently a sequence of four amino acids from the cell-binding domain of fibronectin has been termed the 'cell recognition site' of this multidomain molecule since it mediates cell attachment and inhibits cell adhesion to intact fibronectin. We show here, however, that substrata coated with an isolated cell-binding domain of fibronectin are not sufficient for complete cell adhesion; cells attach and spread but, unlike those adhering to intact fibronectin, they do not form stress fibres terminating in focal adhesions. An additional external stimulus is needed for this cytoskeletal reorganisation and may be provided by one of two heparin-binding fragments of fibronectin. The two 'signals' required for complete adhesion need not be provided simultaneously since focal adhesion formation can be promoted by stimulating cells pre-spread on a cell-binding fragment of fibronectin with a soluble heparin-binding fragment. This second stimulation may involve cell membrane heparan sulphate proteoglycans.

Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements.

Abstract: The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter

Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli.

Abstract: The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e. a cell is either completely fimbriated or bald. This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA. The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA. The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively. The DNA sequence of a 3000-bp region containing the two genes has been determined. The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene. They are highly basic implying that they control the phase switch through interaction at the DNA level.

Integrative transformation of Caenorhabditis elegans

Abstract: A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.

The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens.

Abstract: The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence.

DNA helicase activity of SV40 large tumor antigen.

Abstract: Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.

Multiple sequence motifs are involved in SV40 enhancer function.

Abstract: A systematic mutagenesis of the SV40 enhancer indicates that it spans approximately 100 bp and is composed of at least two distinct DNA domains which exhibit very little enhancing activity on their own. Their association results in a 400-fold enhancement of transcription, virtually irrespective of their relative orientation and, to some extent, of the distance between them. Enhancer activity can also be generated by duplication of either domain. We show also that the activity of each domain is due to the presence of several specific sequence motifs. These motifs are found assorted in different combinations in other viral and cellular enhancers.

The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

Abstract: We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor.

Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones.

Abstract: cDNAs which encode bone gla protein (BGP), an abundant gamma-carboxylated protein of bone, have been cloned from rat and mouse osteosarcoma cell lines. DNA sequence analysis indicates that the cDNAs code for both the 50 (rat) or 46 (mouse) amino acids of the mature proteins and a 49 amino acid leader peptide. The leader peptide of each BGP includes the expected hydrophobic signal sequence and an apparent pro sequence. Although there is no homology between the mature forms of BGP and the gamma-carboxylated clotting factors, we note that there is some homology between their leader peptides. These cDNAs have been used to examine the modulation of BGP mRNA levels by osteoblastic cells in response to hormones. The cDNAs have also allowed isolation of the human BGP gene; analysis of this gene indicates the presence of four exons. Comparison of the exon structure of the BGP gene and the Factor IX (a gamma-carboxylated clotting factor) gene suggests that the exons encoding the part of the leader peptides presumably directing gamma-carboxylation arose from a common ancestral sequence.