Showing papers in "The EMBO Journal in 1983"

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

Abstract: A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322 This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells

Easy identification of cDNA clones.

Abstract: A set of six cloning vectors, pUR 278, 288, 289, 290, 291, 292 is presented. These vectors have the cloning sites, BamHI, SalI, PstI, XbaI and HindIII, in all frames at the 3' end of the lacZ gene. Insertion of cDNA in the proper cloning sites leads to a fusion protein of active beta-galactosidase and the peptide encoded by the cDNA. A simple immunoenzymatic assay can be used to identify clones in such a cDNA library.

Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells.

Abstract: Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.

Chimeric genes as dominant selectable markers in plant cells

Abstract: A chimeric gene was constructed consisting of the promoter of the nopaline synthase gene of Agrobacterium tumefaciens and the structural gene of the aminoglycoside phosphotransferase derived from the transposon Tn5 (APH(3′)II) gene). This chimeric gene was recombined into the T-DNA of Agrobacterium. This plasmid was introduced into plant cells by means of the cocultivation method. Here we discusss the functional expression of the N0S-APH(3′)II gene in the resulting transformed cells.

Sequence of the lacZ gene of Escherichia coli.

Abstract: The nucleotide sequence of the lacZ gene coding for beta-galactosidase (EC 3.2.1.23) in Escherichia coli has been determined. Beta-Galactosidase is predicted to consist of 1023 residues, resulting in a protein with a mol. wt. of 116 353 per subunit. The protein sequence originally determined by Fowler and Zabin was shown to be essentially correct and in an Appendix these authors comment on the discrepancies.

Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members.

Abstract: Two families of fungal mitochondrial introns that include all known sequences have been recognized. These families are now extended to incorporate a plant mitochondrial intron and several introns in chloroplast- and nuclear-encoded rRNA and tRNA precursors. Members of the same family share distinctive sequence stretches and a number of potential RNA secondary structures that would bring these stretches and the intron-exon junctions into relatively close proximity. Using several of these introns which have been extensively studied by either biochemical or genetic means, an attempt is made to integrate the available data into a common picture.

Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia.

Abstract: Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

Abstract: A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells.

Abstract: A plasmid including a mouse immunoglobulin mu gene was transfected into the IgG-secreting human lymphoid line HMy2 and mouse B- and pre-B-cell lines WEHI 231 and 18-81; stably transfected cells were selected. Transfected HMy2 cells synthesized mouse immunoglobulin mu chains as a major secreted protein but the WEHI 231 and 18-81 transfectants transcribed the introduced mu gene at lower levels. In HMy2 transfectants, most of the transcription of the introduced heavy chain gene initiated 40 and 62 bp upstream of the beginning of the VH exon translation start, although a small proportion of transcripts initiating further upstream was detected. WEHI 231 and 18-81 transfectants gave a much higher proportion of upstream initiation. Transient expression of the VH exon was monitored following transfection of mouse myeloma with the VH gene DNA in various plasmid constructs. VH transcription was only observed if the plasmids contained a segment derived from the large VH-CH intron of the immunoglobulin heavy chain locus. This segment, located between JH and switch regions, functioned both downstream of the VH exon and upstream in either orientation. The existence of a transcription enhancer element in this region is therefore proposed.

Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumours.

Abstract: The 15;12 chromosome translocation in murine plasmacytomas and the 8;14 in human Burkitt lymphomas often link the cellular myc oncogene to the locus for constant regions of immunoglobulin heavy chains (CH locus). To clarify how and why c-myc translocation occurs, we have sequenced the mouse and human c-myc genes and correlated c-myc transcription with c-myc rearrangement. Both genes comprise three exons; the second and third encode the myc polypeptide, which is conserved between mammals and birds, particularly in its more basic C-terminal half. Southern blots showed that four of 12 Burkitt lines have c-myc linked near CH switch regions and two near the joining region (JH) locus. Hence, immunoglobulin recombination machinery may participate in translocation, although the common myc breakpoint region around exon 1 does not resemble a switch region. Tumours with breakpoints just 5' to exon 1, or distant from c-myc, had normal c-myc mRNAs of 2.25 and 2.4 kb, which differ at their 5' ends, while tumours with breakpoints within exon 1 or intron 1 had altered c-myc mRNAs (2.1-2.7 kb in Burkitt lines), initiated within intron 1. Both types of mRNAs probably yield the same polypeptide. Since the untranslocated c-myc allele was generally silent, translocation to the CH locus must induce constitutive c-myc expression. The presence of c-myc mRNA in immortal but non-tumorigenic lymphoblastoid cell lines may implicate c-myc in an immortalization step.

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

Abstract: The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells.

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

Abstract: Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha-helical coiled-coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino-terminal region built analogously to non-neuronal intermediate filament proteins should allow a co-polymerization process via the interaction of coiled-coil domains. The extra mass of all triplet proteins is allocated to carboxy-terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.

Isolation of a laminin-binding protein from muscle cell membranes.

Abstract: Skeletal muscle myofibers are each ensheathed by a continuous basal lamina consisting predominantly of type IV collagen, laminin and heparan sulfate proteoglycan. In order to identify laminin-binding components in the muscle cell surface, plasma membranes from mouse thigh muscle and from rat L6 myoblasts were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred samples with 125I-labelled laminin revealed a prominent band of approximate mol. wt. 68 000. A protein of this mol. wt. was isolated by affinity chromatography of muscle cell plasma membranes on laminin-Sepharose. The hydrophobic protein has an apparent mol. wt. of 68 000 and has a high content of serine, glycine and acidic amino acids. After detergent solubilization the purified protein binds to laminin-coated Sepharose beads at a higher rate than to beads coated with either fibronectin or collagen types I and IV. The interaction of the protein, called LB 68, with laminin was also studied after incorporation into synthetic lecithin vesicles. While detergent-solubilized LB 68 bound to 125I-labeled laminin only at lower than physiological ionic strength, liposome-incorporated LB 68 bound to laminin in the absence of detergents under physiological conditions. We propose that this protein is involved in the interaction of myoblasts with laminin substrates and thus may participate in the anchorage of the basal lamina in the plasmalemma of myotubes.

An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex.

Abstract: An improved method for the detection of cellular RNAs in tissue sections has been developed. It involves in situ hybridization of tritium-labeled cloned DNA probes to tissue sections and autoradiography. The method was calibrated by using a cloned DNA probe complementary to transcripts abundant in the midgut cells of Drosophila larvae. The improved method also permitted the detection of these transcripts in sectioned embryos where they are much less abundant. The sensitivity of the method can be approximated by quantifying the signal intensities over the hybridizing embryonic midgut cells relative to the larval midgut cells for which the number of transcripts has been estimated. Based on these calculations we estimate that the method is sensitive enough to detect 100 complementary RNA molecules per cell after 3 days of autoradiographic exposure with a signal-to-noise ratio of 10. The method has been successfully applied to detect transcripts of the homeotic gene Antennapedia. Serial sections allow us to study the spatial pattern of gene expression in the course of development.

Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens.

Abstract: Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

DNA sequence and genome organization of genital human papillomavirus type 6b.

Abstract: The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1) All major open reading frames are located on one DNA strand only Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1 The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames The internal part of E2 completely overlaps with another open reading frame E4 The putative late region contains two large open reading frames L1 and L2 The L1 amino acid sequences are highly conserved among analyzed papillomavirus types By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression

Coronavirus mRNA synthesis involves fusion of non-contiguous sequences.

Abstract: Positive-stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non-contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific 'polymerase jumping' event.

Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila.

Abstract: Homeotic genes are involved in the control of developmental pathways: dominant mutations at the Antennapedia locus of Drosophila, for example, lead to replacement of the antennae on the head of the fly by mesothoracic legs. Using a combination of chromosome walking and jumping, we have cloned a DNA region from Drosophila containing Antennapedia. Five DNA inversion rearrangements which are associated with the Antennapedia mutant phenotype were localized within a 25-kb region. Genomic DNA sequences from this area were used as hybridization probes to screen cDNA libraries prepared from Drosophila embryonic and pupal poly(A)+ RNA. A 2.2-kb cDNA sequence (903) was isolated which appears to derive from at least four non-contiguous chromosomal regions that span 100 kb. It includes the positions of the inversion breakpoints. A second cDNA of 2.9 kb (909) is composed of sequences from at least three chromosomal regions, two of which are similar or identical to sequences contained in the 903 clone but the third is derived from genomic DNA within a putative 903 intron. The unusual size and complexity of this locus are discussed.

Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes.

Abstract: At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence.

Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein

Abstract: A set of monoclonal antibodies to desmin has been isolated from a fusion of mouse myeloma cells with spleen cells from mice immunized with purified porcine desmin. Eleven group I antibodies recognized desmin in the immune blot, and using defined desmin fragments the epitope has been tentatively assigned as lying between residues 325 and 372. When cell lines were tested in immunofluorescence only the human line RD and hamster BHK-21 were positive. When tissue sections were used, skeletal, cardiac, visceral and some vascular smooth muscle cells were positive. Thus, the group I antibodies appear specific for desmin and do not recognize other intermediate filament proteins. Group II monoclonals recognized not only desmin in the immune blot but also other polypeptides. The epitope of this class is located between residues 70 and 280. In immunofluorescence on cell lines and tissues, the staining patterns of group II antibodies were more complicated and demonstrate that not only other intermediate filament proteins but also additional antigenic determinants are being recognized. The group I antibodies stain, as expected from their desmin specificity, rat and human rhabdomyosarcomas and thus appear to be useful reagents in pathology.

The location of Ultrabithorax transcripts in Drosophila tissue sections.

Abstract: I have used in situ hybridization to map the distribution of transcripts of the Ultrabithorax unit in tissue sections of Drosophila embryos and larvae. The results confirm the prediction of Lewis that genes of the bithorax complex will show segmentally regulated patterns of expression. Transcripts of the Ultrabithorax unit are detected in derivatives of the ectoderm and in some derivatives of the mesoderm, but not in the endoderm. In each of these tissues the transcripts are found in the derivatives of some segments but not of others. In third instar larvae they are most abundant in the imaginal discs of the third thoracic segment, and in a region of the central nervous system that includes parts of the metathoracic and first abdominal neuromeres. They are also detected in the nuclei of polytene cells of the larval epidermis, principally in the third thoracic and first abdominal segments, and in the nuclei of larval muscles in the first six abdominal segments. In late stages of embryogenesis the central nervous system is the most prominently labelled tissue; within it transcripts are found only in the neuromeres of the thoracic and abdominal segments. They are most abundant in a region which includes parts of the neuromeres of the metathorax and the first abdominal segment.

The DNA sequence of the H-2Kb gene. Evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibilty antigens

Abstract: We have determined the DNA sequence of the H-2Kb gene of the C57B1/10 mouse. Comparison of this sequence with that of the allelic H-2Kd shows surprisingly that the exons have accumulated more mutations than their introns. Moreover, many of these changes in the exons are clustered in short regions or hot spots. Additional comparison of these sequences with the H-2Ld and H-2Db sequences shows that, in several cases, the altered sequence generated at the hot spot is identical to the corresponding region of a non-allelic H-2 gene. The clustered changes are responsible for 60% of the amino acid differences between the H-2Kb and H-2Kd genes and suggest that micro-gene conversion events occurring within the exons and involving only tens of nucleotides are an important mechanism for the generation of polymorphic differences between natural H-2 alleles.

Evidence for a repeating cross-beta sheet structure in the adenovirus fibre.

Abstract: The amino acid sequence of the adenovirus fibre protein reveals an approximately repeating motif of 15 residues. A diagonal comparison matrix established that these repeats extended from residue 43 to residue 400 of the 581 residue sequence. Assignment of secondary structure combined with model building showed that each 15-residue segment contained two short beta-strands and two beta-bends, one of which incorporated an extra residue in a beta-bulge of the Gx type. The 44 strands together gave a long (210 A) narrow, amphipathic beta-sheet, which could be stabilised by dimer formation to give the shaft of the fibre. The knob could arise from a dimer of the C-terminal 180 residue segment, predicted to be an 8-10 stranded beta-sandwich. This model is consistent with the electron micrographs of the fibre and it was supported by measurements of c.d. and of electron diffraction from microcrystals. The latter gave a pair of wide angle arcs, corresponding to a repeat of 4.7 A, oriented appropriately for a cross-beta structure. The relation of this structure to globular structures is discussed and a folding pathway is proposed. In its general features the structure resembles that proposed for the tail fibre of bacteriophage T4.

Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo.

Abstract: When submitted to a heat-shock, mouse embryonal carcinoma (EC) and fibroblast cells show very different behavior. All the EC cells so far analyzed express very high levels of several heat-shock proteins (HSP) in the absence of stress and independent of their origin and culture conditions. In such cells, the 89-kd, 70-kd and 59-kd HSP are the most prominent proteins after actin. In addition, the 89-kd and 59-kd HSP are not stimulated by an arsenite shock in contrast to what is observed with fibroblasts or cells of the parietal yolk sac type. Arsenite induces the synthesis of a 105-kd polypeptide in fibroblasts but not in EC cells. In vitro differentiation of F9 cells induced by retinoic acid and dibutyryl cAMP is accompanied by a decrease in the spontaneous relative abundance of HSP and restores the arsenite-induced synthesis of the 105-kd polypeptide. EC cells are usually believed to be similar to inner cell mass cells of mouse blastocyst. Furthermore, data in the literature together with our own results suggest that the same three HSP are also spontaneously expressed in high amounts in the early mouse embryo.

Codon-specific missense errors in vivo.

Abstract: We have developed a simple method for measuring the missense substitution of amino acids at specified positions in proteins synthesized in vivo We find that the frequency of cysteine substitution for the single arginine in Escherichia coli ribosomal protein L7/L12 is close to 10(-3) for wild-type bacteria, decreases to 4 x 10(-4) in streptomycin-resistant bacteria containing mutant S12 (rpsL), and is virtually unchanged in Ram bacteria containing mutant S4 (rpsD) We have also found that the frequency of the cysteine substitution for the single tryptophan in E coli ribosomal protein S6 is 3-4 x 10(-3) for wild-type bacteria, decreases to 6 x 10(-4) in streptomycin-resistant bacteria and is elevated to nearly 10(-2) in Ram bacteria

Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor

Abstract: The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle part of neurophysin and exon C its variable C terminus together with glycoprotein. Thus, the three functional domains of the percursor - arginine, vasopressin, neurophysin, glycoprotein - are encoded on three distinct exons.

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

Abstract: From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.

Spatial distribution of Antennapedia transcripts during Drosophila development.

Abstract: We have localized transcripts specified by the homeotic Antennapedia (Antp) locus within serial tissue sections of Drosophila embryos and larvae by in situ hybridization. As a hybridization probe we used a 2.2-kb cDNA sequence (903) which is complementary to at least four non-contiguous chromosomal DNA regions within a span of 100 kb derived from the Antp+ locus. The tritiated probe was directly hybridized to frozen tissue sections of wild-type embryos and larvae. Hybridization was first detected to the progenitors of the thoracic segments during the cellularization of the syncytial blastoderm, when embryonic cells first become determined to form particular adult segments. At later embryonic stages the ventral nervous system also becomes labeled in a spatially-restricted manner. Initially, accumulation of transcripts is detected in all thoracic and abdominal ganglia of the ventral cord. Subsequently, the highest concentration of transcripts is detected in the ganglion cells of the mesothorax. Proper development of this segment is known to be affected in individuals homozygous for putative Antp null alleles. In third-instar larvae, hybridization to the different imaginal disks of all three thoracic segments is observed, localized mainly to those regions that will form proximal, cuticular portions of the respective segments. These results are discussed with respect to the role of homeotic genes in the specification of particular anatomical segments.

Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase genes and a nifA-like gene.

Abstract: The region of the Rhizobium leguminosarum plasmid pRL1JI involved in nodulation and nitrogen fixation has been cloned on a series of four overlapping cosmid clones. These clones represent ˜60 kb of pRL1JI DNA on which a series of Tn5-induced fix and nod alleles have been identified, with the two most distant alleles being separated by ˜45 kb of DNA. The mutant alleles fell into three groups, two clusters of fix alleles separated by one cluster of nod alleles. Within one group of fix alleles, DNA homologous to the nifA gene of Klebsiella pneumoniae has been identified, whereas the pRL1JI DNA homologous to the K. pneumoniae nitrogenase genes is present within the other group of fix alleles.

Coding and 3' non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme.

Abstract: The nucleotide sequence of an almost complete cDNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) has been determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A-rich stretch of the 5' non-coding region and the complete 3' non-coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N-terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent mol. wt. of the subunit of the purified enzyme.