Molecular Cloning and Characterization of the cDNA for Discoidin II of Dictyostelium discoideum

By using monoclonal antibodies directed against discoidin II,we have isolated cDNA clones from axenically grown Ax-2 cells.On cDNA clone (D2) condtained a 1.2-k.b insert encoding theentire discoidin II protein, which is conposed of 257 aminoacid residuces and has a calculated molecular mass of 28,574.The amino acid sequences, determined by Edman degradation ofsix tryptic peptides of discoidin II, were identical to thosededuced from the cDNA sequences. The protein bears no resemblanceto any proteins in the data banks, except that its sequenceis 49% identical with the amino acid sequence of discoidin I.Discoidin II shares with discoidin I both a carbohydratebindingsite and an Arg-Gly-Asp (RGD) sequence, which has been foundin fibronectin in mammalian cells. With the onset of aggregation(8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate.A similar pattern of regulation occurs at the protein level. ¹Present address: MRC Laboratory for Molecular Cell Biology,University College London, Gower Street, London, WC1E 6BT UnitedKingdom     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum

The distribution of discoidin I and discoidin II, developmentally regulated lectins in Dictyostelium discoideum, was determined immunohistochemically at various stages of development. Discoidin I was first prominent as focal clumps in aggregating cells, then accumulated on the surface of aggregates and around them. Discoidin II became prominent later and ultimately localized in what appear to be prespore vesicles. The results indicate that discoidin I and discoidin II have different and possibly multiple functions.     

Receptor for the cell binding site of discoidin I

Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.     

Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family

We analyzed five malate synthase cDNA clones from the higher plant Brassica napus L. We determined the complete mRNA sequence and showed that the longest cDNA clone, pMS1, contains the entire protein coding region. The deduced polypeptide consists of 561 amino acids with a molecular mass of 63,700 daltons. To discern whether the cloned mRNAs represent distinct malate synthase polypeptides, we compared restriction maps and partial nucleotide sequence of the cDNA clones as well as their pattern of hybridization with restriction fragments in nuclear DNA. The results suggest that the five cloned mRNAs are encoded by either a single gene or by highly conserved members of the gene family.     

Detailed analysis of the portion of the herpes simplex virus type 1 genome encoding glycoprotein C.

We previously showed that the right third of HindIII fragment L (0.59 to 0.65) of herpes simplex virus type 1 (HSV-1) encodes a family of mRNAs some members of which appear to be related by splicing. In the experiments described in this communication, we determined the nucleotide sequence of the DNA encoding this mRNA family and precisely located the mRNAs associated with this DNA sequence. The major mRNA species is unspliced and encoded by a 2.520-nucleotide region. Just upstream of the 5' end are TATA and CAT box sequences characteristic of HSV-1 promoters. The 3' end maps near a region containing a nominal polyadenylation signal. Three minor species (2,400, 2,200, and 1,900 bases, respectively) appear to share a very short leader sequence with the 5' end of the major mRNA and are then encoded by uninterrupted DNA sequences beginning about 100, 400, and 625 bases downstream of the 5' end of the major unspliced mRNA. These positions map at or very near positions which agree reasonably well with consensus splice acceptor sequences. The fourth mRNA is encoded by a contiguous 730-nucleotide sequence at the 3' end of the major unspliced mRNA and has its 5' end just downstream of recognizable TATA and CAT box sequences. We suggest that this mRNA is controlled by its own promoter. The nucleotide sequence data, in combination with the mRNA localization, demonstrate four potential polypeptides encoded by the region. The largest is 1,569 bases long and defines a 523-amino acid protein with sequence features characteristic of a glycoprotein. This was confirmed to be HSV-1 glycoprotein C by immune precipitation of the in vitro translation product of the major unspliced mRNA, performed with a polyspecific antibody to HSV-1 envelope glycoproteins (anti-env-1 serum), and by comparison of tryptic peptides of this translation product with those of authentic HSV-1 glycoprotein C. Polypeptides encoded by some of the minor species also were tentatively identified.     

70-Kilodalton heat shock polypeptides from rainbow trout: characterization of cDNA sequences.

RTG-2 cells, a line of fibroblasts from rainbow trout (Salmo gairdnerii), are induced to synthesize a distinct set of heat-shock polypeptides after exposure to elevated temperature or to low concentrations of sodium arsenite. We isolated and characterized two cDNA sequences, THS70.7 and THS70.14, encoding partial information for two distinct species of 70-kilodalton heat shock polypeptide (hsp70) from these cells. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with derived amino acid sequences for hsp70 polypeptides from Drosophila melanogaster and Saccharomyces cerevisiae. Northern blot analysis of RNA from arsenite-induced RTG-2 cells, with the trout hsp70 cDNAs as probes, revealed the presence of three hsp70 mRNA species. Southern blot analysis of trout testis DNA cleaved with various restriction endonucleases revealed a small number of bands hybridizing to the hsp70 cDNAs, suggesting the existence of a small family of hsp70 genes in this species. Finally, trout hsp70 cDNA sequences cross-hybridized with restriction fragments in genomic DNA from HeLa cells, bovine liver, Caenorhabditis elegans, and D. melanogaster.     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA.

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.     

Genes encoding proteins with homologous contiguous repeat sequences are highly expressed in the serous cells of the rat submandibular gland

An abundant class of secreted salivary polypeptides is characterized by the presence of identical and contiguous repeats of amino acid sequences within the polypeptide chains, and includes the proline-rich proteins. We discovered a new family of contiguous repeat polypeptides (CRPs) that is related to the proline-rich proteins but contains little proline. Analysis of salivary mRNAs and liver DNA by molecular cloning, DNA sequence determinations, and Northern and Southern blot hybridization revealed several closely related CRP mRNAs and at least 10 CRP-related genes. We further analyzed two CRP mRNAs of 850 and 920 nucleotides and the gene encoding the larger CRP mRNA. The two mRNAs contain the same 69-base repeats in their coding regions and are identical in their 5'- and 3'-untranslated tracts. However, they differ in the number of contiguous repeats (four versus five) and a segment at the 3' end of the coding region which encodes closely related but unique COOH termini of the CRPs. These structural features suggest a recent gene conversion. The CRP gene analyzed is divided into three exons that encode (i) 5'-untranslated tract and signal sequence, (ii) secreted polypeptide, and (iii) 3'-untranslated tract, respectively. CRP mRNA contains two open reading frames. The longer open reading frame encodes a CRP precursor with a signal sequence of 17 amino acids, four to five contiguous repeats of 23 amino acids, and a variable COOH region that begins with two segments related to the contiguous repeats. Immunochemical analysis of salivary gland slices with antisera raised against peptides corresponding to two regions of the larger open reading frame revealed intense staining only of the serous cells of the submandibular glands. 35S-Labeled oligonucleotides complementary to CRP mRNA specifically hybridized to the same cells.     

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.     

Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley

We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.