A novel gene—samfR involved in early stage ofStreptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation.     

Internal Changes in Hyphae of Rhizopus sexualis (Smith) Callen and Mucor hiemalis Wehm. associated with Zygospore Formation

The distribution of nucleic acids, nuclei, mitochondria, andreserve foods in vegetative hyphae, zygophores, and developingzygospores of Rhizopus sexualis and Mucor hiemalis were examinedby differential staining. The extreme tips and growing zones of vegetative hyphae containeda high concentration of RNA and numerous mitochondria. Nucleiwere not present at the extreme tip but were numerous just behindit. In older parts of the hyphae the concentration of RNA waslow and both nuclei and mitochondria were fewer than in thezone of elongation. Glycogen and lipids were present in all parts of the livinghyphae except the extreme tips and were more highly concentratedin the older parts of the hyphae. Young zygophores showed a much lower RNA/DNA ratio than thatfound in the vegetative hyphal tips. Transfer of colonies from20? C to temperatures of less than 10? C, which is known toprevent zygospore initiation, caused some but not all recognizablezygophores of R. sexualis, but not those of M. hiemalis, torevert to the RNA/DNA ratio characteristic of vegetative hyphae.Some zygophores of Rhizopus and most of those of Mucor developedinto sporangiophores at low temperature, retaining the relativelylow RNA/DNA ratio throughout development. It is suggested thata reduction in the RNA/DNA ratio is an early step in the changefrom the vegetative state to the reproductive one. At firstthis step is reversible, but soon becomes irreversible by anadditional step, the nature of which is unknown. For some timeafter this the reproductive hyphae are capable of either producingasexual sporangia or of conjugating to produce zygospores. Onceconjugation has taken place development either ceases or continuesuntil the spore is fully mature, but it cannot under any circumstancesthen be reversed. The development and maturation of the zygospore involves a greatincrease in number of both nuclei and mitochondria and in theconcentration of glycogen and lipids.     

Identification of the domain of Saccharomyces cerevisiae adenylate cyclase associated with the regulatory function of RAS products

Summary Various truncated CYR1 genes of Saccharomyces cerevisiae were fused to efficient promoters and expressed in Escherichia coli and S. cerevisiae cells with or without the RAS genes. The catalytic domain of adenylate cyclase encoded by the 3-terminal 1.3 kb region of the open reading frame of the CYR1 gene produced cyclic AMP, irrespective of the presence of RAS genes. The product of the 3-terminal 2.1 kb region of CYR1 showed guanine nucleotidedependent adenylate cyclase activity and produced a large amount of cAMP in the presence of the RAS gene. Thus, the domain encoded by the 0.8 kb region adjacent to the catalytic domain is associated with the regulatory function of the RAS products. The cyr1 RAS1 RAS2 cells carrying the 3-terminal 1.3 kb region of CYR1 were unable to respond to environmental signals such as sulfur starvation and temperature shift, but the cyr1 cells carrying the 2.1 kb region and at least one RAS gene were able to respond to these signals. The environmental signals may be transferred to the adenylate cyclase system through the RAS products.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation. Project supported by the National Natural Science Foundation of China (Grant No. 39830010).     

Developmental gene regulation in Aspergillus nidulans

The Ascomycete fungus Aspergillus nidulans reproduces asexually by differentiating conidiophores and conidia. Gene regulation during asexual reproduction was investigated by comparing poly(A) RNA populations derived from somatic hyphae, conidiating cultures and purified conidia. Single-copy and complementary DNA hybridization experiments showed that vegetative cells contained 5600–6000 diverse, average-sized poly(A) RNA sequences distributed into three prevalence classes. cDNA hybridization experiments indicated that a significant proportion of the poly(A) RNA derived from either conidiating cultures or spores consisted of sequences absent from somatic hyphae. To assess accurately the degree to which the poly(A) RNA populations differed, cDNA preparations were isolated which were complementary to sequences present only in conidia or in conidiating cultures. Hybridization of these cDNAs with poly(A) RNA from conidiating cultures showed that approximately 18.5% of the poly(A) RNA mass comprised 1300 diverse sequences not present in somatic cells. Of these, about 300 were present only in conidia. The remainder were accumulated specifically during sporulation, but were absent from spores. Analogous experiments showed that the great majority of the poly(A) RNA sequences accumulated by vegetative hyphae were also present in conidiating cultures. Thus, cell differentiation during A. nidulans asexual reproduction involves the accumulation of many new poly(A) RNA sequences, but not the loss of preexisting ones.     

Nitrogen fixation genes (nif K,D,H) in the filamentous nonheterocystous cyanobacteriumPlectonema boryanum do not rearrange

The organisation of the structural genes for nitrogen fixation (nif K,D and H) in a nonheterocystous, filamentous cyanobacteriumPlectonema boryanum has been examined in comparison with a heterocystous cyanobacterium,Anabaena torulosa. DNA from repressed (fix-) cultures ofA. torulosa showed a discontinuousnif region spread over approximately 18 kb, an arrangement typical of the vegetative cells of heterocystous cyanobacteria. The region contained a contiguousnif DH separated fromnif K. by nearly 11 kb DNA. The intervening 11 kb DNA harboured the genexis A involved in the rearrangement ofnif K,D,H to form a cluster during differentiation of heterocysts. DNA fromPlectonema boryanum had a small, contiguousnif KDH cluster spanning a region of approximately 4 kb. DNA homologous to the 11 kb excison with its residentxis A was not present.Nif hybridisation patterns of restriction digests of the DNA isolated from repressed (fix^-) or induced (fix^--) cultures ofP. boryanum were completely identical. These results unequivocally demonstrate that in the nonheterocystous cyanobacterium, unlike in the heterocystous strains, no gene rearrangement, either within thenif KDII cluster or in its vicinity, accompanies the expression of nitrogenase activity.     

Internuclear Genetic Transfer in Vegetative Dikaryons of SCHIZOPHYLLUM COMMUNE: I. Di-Mon Mating Analysis

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd ⁺ and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd⁺ nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd ⁺ allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd⁺ + mnd] cells, but none was found to be entirely satisfactory.     

A Medium-Copy-Number Plasmid for Insertional Mutagenesis ofStreptococcus mutans

We have constructed a plasmid useful for insertional mutagenesis inStreptococcus mutans.The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates inEscherichia coli,does not replicate inS. mutans,contains an erythromycin-resistance marker which can be selected inE. colior the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library ofS. mutansDNA inE. coliand show that the clones can be reintegrated into theS. mutanschromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of theE. coli drpAgene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked todrpAinS. mutanswith high homology to IS861.     

Molecular Cloning of the Fujinami Sarcoma Virus Genome and Its Comparison with Sequences of Other Related Transforming Viruses

Full-length proviral DNA of Fujinami sarcoma virus (FSV) of chickens was molecularly cloned and characterized. An analysis of FSV DNA integrated in mammalian cells showed that restriction endonuclease SacI has a single cleavage site on FSV DNA. Unintegrated closed circular FSV DNA obtained from newly infected cells was linearized by digestion with SacI and cloned into λgtWES·λB. The following three different molecules were isolated: FSV-1 (4.4 kilobases [kb]) and FSV-2 (4.7 kb), which appeared to be full-length FSV DNA molecules containing either one or two copies of the long terminal repeat structure, and FSV-3 (6 kb), which consisted of part FSV DNA and part DNA of unknown origin. An analysis of the structure of cloned FSV-1 and FSV-2 DNA molecules by restriction endonuclease mapping and hybridization with appropriate probes showed that about 2.6 kb of the FSV-unique sequence called FSV-fps is located in the middle of the FSV genome and is flanked by helper virus-derived sequences of about 1.3 kb at the 5′ end and 0.5 kb at the 3′ end. The long terminal repeats of FSV were found to have no cleavage site for either EcoRI or PvuI. Upon transfection, both FSV-1 DNA and FSV-2 DNA were able to transform mammalian fibroblasts. Four ³²P-labeled DNA fragments derived from different portions of the FSV-fps sequence were used for hybridization to viral RNAs. We found that sequences within the 3′ half of the FSV-fps gene are homologous to RNAs of PRCII avian sarcoma virus and the Snyder-Theilen strain of feline sarcoma virus, both of which were previously shown to contain transforming genes related to FSV-fps. These results suggest that the 3′ portion of the FSV-fps sequence may be crucial for the transforming activity of fps-related oncogenic sequences.     

The azurin gene from Pseudomonas aeruginosa codes for a pre-protein with a signal peptide: Cloning and sequencing of the azurin gene

The azurin gene from Pseudomonas aeruginosa is located on a 1.3 kb long PstI DNA fragment. Its nucleotide sequence has been determined. It appears that the gene codes for a pre-protein with a 19 amino acid long signal sequence which possibly assists in the transport of the azurin over the periplasmic membrane.     

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, ψnad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this ψnad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

Sequence arrangements of the Escherichia coli chromosome and of putative insertion sequences, as revealed by electron microscopic heteroduplex studies.

Total Escherichia coli DNA from strain DK445 (which is CSH50 F^?, R^?, deletion lac and pro, lysogenized with lambda cIts857 Sam7 lac5: :Mu cI⁺) was denatured, reannealed, and observed by electron microscopy. The single-strand DNA lengths ranged from about 50 to 150 kilobases (kb). In some molecules a short duplex region with a single-stranded fork at each end was observed. The duplex lengths were 0.75 kb, 1.30 kb, 5.22 kb, 5.62 kb, which correspond to those of IS1; of IS2, IS3, or IS4; of the ribosomal RNA genes; and of the γδ sequence, respectively. Duplexes of 1.0 kb and 0.5 kb were also found. Most of the duplexes of 0.5, 0.75, 1.0 and 1.3 kb were observed as intramolecular stem-loop structures and were therefore interpreted to be sequence duplications in inverted order on the same DNA strand. The most frequent separations of the putative inverted insertion sequences were around 22 and 27.5 ± 1.5 kb. About 14% of the E. coli chromosome is estimated to be involved in the sequence arrangements that give rise to stem-loop structures upon denaturation and reannealing. The copy numbers of the putative insertion sequences and other elements that form the “stems” of the stem-loop structures are also estimated.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.