Pro Gene Research Articles

Cellular basis of the effects of gibberellin and the pro gene on stem growth in tomato

Tomato (Lycopersicon esculentum Mill.) plants homozygous for the mutant pro gene, exhibiting the distinctive procera phenotype, appeared virtually identical to gibberellic acid (GA3)-treated isogenic normal plants. The pro gene and GA3 caused analogous increases in internode length, and in the length and number of cells in the outer cell layers of each internode. Internode number was also increased by pro and GA3 over the period of the experiment. Despite their greater length, the internodes of GA3-treated and pro plants reached their final size within a time period similar to that of internodes of untreated normal plants. The pro mutant itself was responsive to GA3, especially in the seedling stage, but the proportional increase in height seen in the later stages of growth was less than that of normal plants.

Multiple sequence rearrangements accompanying the duplication of a tRNAPro gene in wheat mitochondrial DNA

In the course of isolating tRNA genes from wheat mtDNA, we have found the same tRNA(Pro) gene in two different Hind III restriction fragments, H-P1 (0.7 kbp) and H-P2 (1.7 kbp). Sequences immediately flanking these duplicate genes are closely related, although not identical; sequence comparisons suggest that multiple rearrangements have occurred in the vicinity of the H-P2 tRNA(Pro) gene, relative to the H-P1 version. The chimeric nature of H-P2 is emphasized by the presence of sequences that are also found upstream of the wheat mitochondrial 26S rRNA gene, as well as sequences derived from chloroplast DNA. Comparison of H-P2 with H-P1 plus upstream sequences provides some insight into possible molecular events that might have generated H-P2. In particular, such comparisons suggest a model in which the homologous sequences in H-P2 are seen to be derived from H-P1 plus upstream sequences as a result of an intragenomic, site-specific rearrangement event, followed by amplification of the product, its fixation in the mitochondrial genome, and subsequent sequence divergence (single base changes as well as insertions/deletions of up to 50 nucleotides). The results reported here implicate particular primary sequence motifs in certain of the rearrangements that characterize H-P2.

RNA polymerase III promoter elements enhance transcription of RNA polymerase II genes.

Using transient expression assays in cultured human cells we have observed that RNA Polymerase III promoter sequences exert a positive cis-acting enhancer effect on RNA Polymerase II transcription. A DNA segment containing a copy of the Alu repeated element enhances transcription of the liver specific Haptoglobin related (Hpr) promoter in Hepatoma cell lines but not in HeLa cells. A tRNA(pro) gene acts as enhancer of the SV40 promoter both in Hepatoma and in HeLa cell lines. Transcription from the SV40 promoter is also enhanced by DNA segments containing only the box A or the box B of the tRNA(pro) promoter.

Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA.

We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNA(Thr) and tRNA(Pro) genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence of unique mechanisms for the regulation of gene expression in this organism.

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Homology in the region containing a tRNA(Trp) gene and a (complete or partial) tRNA(Pro) gene in wheat mitochondrial and chloroplast genomes.

We have used bean mitochondrial (mt) and chloroplast (cp) tRNA(Trp) as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNA(Trp) genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA(Trp) genes are 97% homologous. On the wheat cp DNA, a tRNA(UGGPro) gene was found 139 bp upstream of the cp tRNA(Trp) gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA(Pro) gene was found 136 bp upstream of the mt tRNA(Trp) gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA(Trp) gene separated by about 140 bp from a tRNA(Pro) gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA(Trp) gene and the tRNA(Pro) (or partial tRNA(Pro)) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence. Wheat mt tRNA(Trp) gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria.

Genes that cooperate with tumor promoters in transformation.

Tumor-promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro-1 confers lifespan extension on these cells. Recently, we have found that a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced synthesis of two proteins of 15 and 16 kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation.

Lifespan extension of basal cell nevus syndrome fibroblasts by transfection with mouse pro or v-myc genes.

Dermal fibroblasts from patients with the autosomal dominant cancer-prone disease Basal Cell Nevus Syndrome (BCNS) exhibit a serum dependence, anchorage dependence and in vitro lifespan (about 20 population doublings or less) similar to those of fibroblasts from normal age-, race- and sex-matched controls. Transfection with v-myc or with an activated mouse pro-I gene (which specifies sensitivity to promotion of neoplastic transformation in JB6 mouse epidermal cells) specifically conferred partial immortality on the BCNS fibroblasts by substantially extending their population doubling levels by more than 19 population doublings. This suggests that either v-myc or pro-I gene can cooperate with BCNS gene(s) to produce an extension of lifespan or partial immortality. However, the transfected BCNS fibroblasts that escaped senescence were anchorage-dependent even after exposure to the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate (TPA), epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). These observations indicate that BCNS fibroblasts differ from their normal counterparts in susceptibility to extended growth and may therefore be pre-neoplastic. It is clear that they require more than an activated pro or myc gene for progression to the tumor cell phenotype.

Localization, sequence and expression of the gene coding for tRNAPro (UGG) in plant mitochondria

The four Sal I fragments of wheat mitochondrial DNA containing the 18S and 5S ribosomal RNA genes were screened for the presence of tRNA genes. Upon sequencing, a tRNA(Pro) (UGG) gene was found in two of these four fragments. The localization of the corresponding gene on the maize mitochondrial genome was established. Transcriptional studies have shown that this gene is transcribed in wheat and maize mitochondria. The sequence of the corresponding tRNA(Pro) (UGG) of bean mitochondria was determined using in vitro post-labeling techniques.

DNA sequences in human nasopharyngeal carcinoma cells that specify susceptibility to tumor promoter-induced neoplastic transformation.

Homologs of the recently described mouse pro genes, that transfer sensitivity to promotion of neoplastic transformation by tumor promoters, have been cloned from a genomic library of the human nasopharyngeal carcinoma (NPC) cell line CNE2. Both pro-1 and pro-2 homologs were identified by screening this library with mouse probes, but only the pro-1 homologs were able to confer sensitivity to TPA-induced transformation when transferred to promotion-insensitive mouse JB6 cells. This suggests a possible role for the putative pro-1 gene in the etiology of human NPC.

Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis.

Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile). The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete. A majority of the tRNA genes are located in close proximity to one another. A unique feature of the Pro and Ile genes is that their DNA sequence overlap.

Common and interchangeable elements in the promoters of genes transcribed by RNA polymerase III

We have shown that the 34 bp internal control region of the somatic 5S RNA gene from Xenopus borealis can be split into two separable components. A maxigene carrying an insertion between nucleotide 71 and nucleotide 74 of the coding region is actively transcribed in the nucleus of X. laevis oocytes, giving rise to a maxitranscript with initiation and termination points identical with those of the wild-type transcript. The first 11 bases of the 5S RNA gene promoter are shown to be structurally and functionally homologous with the first component (box A) of the promoter for tRNA genes. This was shown by constructing hybrid 5S RNA-tRNA Pro and tRNA Pro-5S RNA genes that were efficiently transcribed in the X. laevis oocytes. Initiation of transcription appears to be a complex phenomenon in which both components of the internal promoter play a role.

Transduction of Escherichia coli trp genes in Salmonella typhimurium and effect of N-methyl-N'-nitro-N-nitrosoguanidine on transduction with heterogenotic DNA.

P1Kc-mediated transduction of Escherichia coli trp genes occurred at a frequency of about 10(-8) in Salmonella typhimurium trp strains carrying mutations determining sensitivity to P1 and a low level of restriction enzymes. Heterospecific transductants were analysed by using them as donors in second-stage transductions mediated by bacteriophage KBint. One class of heterospecific transductants had the phenotype Trp+ Pro- but were extremely unstable and reverted at high frequency (up to 80%) to the parental phenotype. The Trp+ Pro- phenotype probably represents insertions of the E. coli trp genes in the S. typhimurium pro genes. It was stable in a RecA background. Haploid Pl-mediated heterogenotes exhibited various degrees of homology with the trp region of S. typhimurium in KB-mediated second-stage transductions: one heterogenote (MA427), which transduced the linked markers trp, cysB and pyrF very poorly, was used to look at the effects of the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in stimulating heterologous recombination and to derive a method for positive selection of linked mutations. Following treatment with MNNG, heterospecific Trp+ transductants were obtained with MA427 as donor. Their yield was maximal immediately after MNNG treatment but decreased and even disappeared when transductions were carried out a few hours later. This transient process is thought to represent MNNG-induced conversion of abortive transductants to complete transductants. On the other hand, phenotypic analysis of MNNG-induced Trp+ heterologous transductants revealed the presence of mutations of various phenotypes. In particular, the antimutator phenotype was recognized in 15 clones among 110 MNNG-induced recombinants tested. However, most of these mutations seemed not to influence heterologous recombination.

Nucleotide sequence and evolution of the rat mitochondrial cytochrome b gene containing the ochre termination codon

The nucleotide sequences of the genes for cytochrome b and three potential transfer RNAs (tRNA Pro, tRNA Thr and tRNA Glu) in cloned rat mitochondrial DNA were determined. The derived amino acid sequence of the cytochrome b protein from the light strand indicated that the C-terminal amino acid is asparagine and the ochre termination codon is encoded in the DNA, in contrast to the lack of termination codon in the reading frame of human [Anderson et al., Nature 290 (1981) 457] or mouse [Bibb et al., Cell 26 (1981) 167] mitochondrial DNA. The first ATG codon of the cytochrome b gene was spaced five nucleotides from the 5′-end of the tRNA Glu gene on the heavy strand. There was a single nucleotide spacing between the termination codon of the cytochrome b gene and the 5′ end of the tRNA Thr gene in the light strand. There was also a single nucleotide spacing between the 3′-end of the tRNA Thr gene and the 3′-end of the tRNA Pro gene on the heavy strand. The amino acid and nucleotide sequences of the cytochrome b genes of mammals and yeast [Nobrega and Tzagoloff J. Biol. Chem. 255 (1980) 9828] were compared to reveal structural differences in two very different species. At the same time, amino acid substitutions in particular regions of the mammalian gene corresponding to the exon-intron boundaries in the yeast gene were noted. These genetic features are discussed in relation to the extreme compression of genetic information in the mammalian mitochondrial genome as related to the evolution of the gene organization and its sequence.

Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage.

Heat-resistant derivatives of a Bacillus subtilis 168 strain carrying an xhi mutation, which causes heat-sensitive induction of the PBSX prophage, have been isolated and screened for the acquisition of auxotrophy. Two classes of auxotrophs were isolated, namely Pro- and Pro-Met-; they lacked the ability to produce PBSX, as shown by their resistance to mitomycin C-induced lysis. The proline and methionine requirements and the resistance to mitomycin C were shown to segregate together in phage PBS1-mediated transduction crosses and to be linked to thiB, which is known to be co-transducible with the PBSX prophage. It was therefore proposed that these strains had deletions which removed all or part of the PBSX prophage together with adjacent bacterial DNA encoding the pro(AB) and metC genes. The met mutation was shown to be metC in PBS1 transduction crosses; this gene is known to be co-transducible with the PBSX prophage. The proline requirement was probably due to the deletion of a pro gene which was demonstrated to lie between the PBSX prophage and metC and which was 90% co-transducible with metC. These deletions have been transduced into a strain which was cured of phage SP beta, another bacteriophage carried by B. subtilis 168. No phage particles could be seen in mitomycin C-induced cultures of such strains. The PBSX-deletion strains grew with the same generation time as the PBSX+ parent in L-broth (27 min at 35 degrees C) but they were slower in minimal medium (e.g. 72 min as against 51 min in the PBSX+ strain). Besides being resistant to mitomycin C-induced lysis, the deletion strains were also resistant to lysis induced by thymine starvation of thymine auxotrophs and the loss of viability of these strains after thymine starvation was 100-fold less than in the PBSX+ parent. The deletion strains had not, however, lost the bacterial autolytic enzymes, since they were still susceptible to lysis when placed under semi-anaerobic conditions.

Analysis of the size alleles of the Pro gene in maize — Evidence for a mutant protein processor

Two major proteins in the maize embryo are specified by gene Pro on the long arm of chromosome 1. The gene is polymorphic and size alleles have been found which produce proteins of different molecular weights as well as CRM- null alleles. Evidence is presented for a protein processor which is specific for the PRO proteins. The processor gene, Mep has been mapped to the long arm of chromosome 5.

Specialized transduction of pro genes by coliphage P1: Structure of a partly diploid P1- pro prophage

Bacteria carrying P1- pro hybrid prophages have been isolated as rare (10— 9Pro + per pfu) Pro + transductants of recA proA Escherichia coli. The proA gene is inserted into the P1 prophage since the transductants become Pro − when the resident P1 is lost. Furthermore, when induced, these recA transductants produce HFT pro lysates which contain defective pro-transducing particles as well as plaque-forming particles which do not transduce pro. The number of PFUs obtained is about one-tenth the number obtained from normal lysogens. The density of the transducing particles was not distinguishable from that of the PFUs. The Pro + transductants obtained by use of these HFT pro lysates (at low m.o.i.) carry only a small segment of the P1 genome, including cam and gene 2, and are not P1-immune. The number of Pro + transductants increases about 20-fold when the recipient contains a helper P1 either as superinfecting phage or as prophage, The Pro + transductants that arise in the presence of helper P1 are P1- pro lysogens formed by recombination between the DNA of the helper P1 and of the transducing particles. Like the original P1- pro lysogens, they produce lysates which contain plaque-forming and pro transducing particles. The circular P1- pro prophage is inferred to be oversized (at least as large in size as a P1 headful) and to have duplicate cam-gene 2 regions, one copy at each junction of P1 and Pro DNA. The DNA packaged after induction of P1- pro lysogens is not terminally redundant. However, after injection into recipient bacteria, recombination can occur between the redundant cam-gene 2 regions giving rise to either circular pro-cam-gene 2 transducing DNA or to circular P1 plaque-forming DNA. Evidence for this partly diploid prophage is the construction of P1- pro prophages which are heterozygous for gene 2.

Phage P1 cryptic: II. Location and regulation of prophage genes

Deletion mapping of a P1 cryptic (P1 cry) lysogen shows that cam r , a marker derived from the R factor parent of P1 cry, is closer to the bacterial pro and lac genes than is any other P1 marker. The isolation of a new HFT phage, λp cam, confirms this order. The fact that cam r is terminal in the prophage suggests that the R factor part of the P1 cry genome is responsible for the integration of P1 cry into the host chromosome. The deletions isolated also indicate that the P1 host restriction gene maps near c2 and c3. A double lysogen carrying P1 cry and a thermally inducible P1 remains thermally inducible. Therefore, the lack of expression of gene c1 + of P1 cry is not caused by the absence of a P1 gene producing a diffusible product needed to turn on c1; it appears to be caused, instead, by loss of or damage to the promoter for c1.