Isoptericola chiayiensis sp. nov., isolated from mangrove soil.

A novel actinomycete, designated strain 06182M-1(T), was isolated from a mangrove soil sample collected from Chiayi County in Taiwan. Phylogenetic analysis based on 16S rRNA gene sequences revealed levels of similarity of 97.0-98.8 % to the type strains of recognized species of the genus Isoptericola. Chemotaxonomic data also supported the placement of strain 06182M-1(T) within the genus Isoptericola. However, the low levels of DNA-DNA relatedness between the novel strain and the type strains of recognized species of the genus Isoptericola, in combination with differential phenotypic data, demonstrate that strain 06182M-1(T) represents a novel species of the genus Isoptericola, for which the name Isoptericola chiayiensis sp. nov. is proposed. The type strain is 06182M-1(T) ( = BCRC 16888(T)  = KCTC 19740(T)).

nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

[Studies on embryonal development of lamina cribrosa of human eyes].

The lamina cribrosa of 291 eyes were studied including 260 eyes taken from embryos and fetus of 7 weeks to full term and 31 eyes from infants under 1 year of age. From the observations, it is discovered that the development of the lamina cribrosa lags behind the development of the optic nerve. In the eyes taken from embryos or fetus < 4 months, only the ectodermal primordium of the lamina cribrosa is seen. The initial scleral part of the lamina cribrosa begins to be formed in the 5th month fetus. The fibrous tissue originating from the choroid and optic nerve sheath contributes the formation of the lamina cribrosa in the 8th month fetus. At this time, the morphology of the lamina cribrosa is similar to that of an adult, but the thickness of the structure is still increasing till one year after birth.

Herbidospora yilanensis sp. nov. and Herbidospora daliensis sp. nov., from sediment.

Two actinomycete strains, 0351M-12(T) and 0385M-1(T), were isolated from sediment samples collected from Yilan County and Dali City in Taiwan, respectively. The two isolates displayed characteristics which were similar to those of the genus Herbidospora. They both produced branched and unfragmented substrate mycelia, and aerial hyphae were not produced on Bennett's, glucose-asparagine, Hickey-Tresner, sucrose-nitrate, yeast extract-malt extract, or inorganic salts-starch agars. Short spore-chains were borne on the tips of sporophores arising from the substrate mycelia on oatmeal and oatmeal-nitrate agars. The spore chains contained 10-20 non-motile, smooth-surfaced, oval spores. Galactose, glucose, mannose and madurose were the whole-cell sugars in both strains and meso-diaminopimelic acid was present in their peptidoglycans. The phospholipid pattern was PIV. The predominant menaquinone was MK-10(H(4)) in both strains. Mycolic acids were not detected. Major cellular fatty acids were iso-C(16 : 0) and 10-methyl C(17 : 0). The DNA G+C contents of the two new isolates were 70.6 mol% (0351M-12(T)) and 70.7 mol% (0385M-1(T)), respectively. The relatedness between the isolates and Herbidospora cretacea NBRC 15474(T) was 40.7-48.5 %. The value of DNA-DNA hybridization between strain 0351M-12(T) and strain 0385M-1(T) was 53.6-54.9 %. These two isolates could also be distinguished from each other and from H. cretacea NBRC 15474(T) by differences in several phenotypic characteristics. We therefore propose the names Herbidospora yilanensis sp. nov. and Herbidospora daliensis sp. nov. for these novel bacteria, with strains 0351M-12(T) (=FIRDI 003(T) =BCRC 16875(T) =LMG 24337(T)) and 0385M-1(T) (=FIRDI 004(T) =BCRC 16876(T) =LMG 24336(T)) as the type strains, respectively.

Actinomadura miaoliensis sp. nov., a thermotolerant polyester-degrading actinomycete.

An actinomycete, strain BC 44T-5(T), with the ability to degrade poly(d-3-hydroxybutyrate) was isolated from a soil sample collected from Miaoli County, Taiwan. The isolate displayed substrate mycelia and short spore chains were borne on the aerial mycelia. Spores were non-motile, round, 1 mum in diameter and spiny. The aerial spore mass was blue. Strain BC 44T-5(T) had meso-diaminopimelic acid as the diagnostic diamino acid of the cell-wall peptidoglycan. Whole-cell sugars of the novel strain were identified as glucose, galactose and madurose. Diphosphatidylglycerol and phosphatidylinositol were detected. The predominant menaquinones were MK-9(H(4)) and MK-9(H(2)). Mycolic acids were not detected. Major cellular fatty acids were iso-C(16 : 0) (14.82 %), C(16 : 0) (14.63 %), C(17 : 0) (13.79 %) and 10-methyl-C(17 : 0) (23.77 %.) The DNA G+C content of strain BC 44T-5(T) was 70.6 mol%. On the basis of phenotypic and genotypic data, it is proposed that strain BC 44T-5(T) (=FIRDI 002(T)=BCRC 16873(T)=LMG 24335(T)) should be classified as the type strain of a novel species of the genus Actinomadura, Actinomadura miaoliensis sp. nov.

Molecular characterization of mutations of nit-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa.

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number, growth rate and enzyme activity.

nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutamine-rich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.

Differentiation of Aspergillus parasiticus from Aspergillus sojae by random amplification of polymorphic DNA.

Aspergillus parasiticus and Aspergillus sojae are two morphologically similar species belonging to the Aspergillus section flavi. A new method to distinguish the two species, the aflatoxin producer A. parasiticus and the koji mold A. sojae, was developed. Single primers with arbitrary sequences were used to generate random amplified polymorphic DNA (RAPD) markers from strains of these two species. Three decamers, OPA-04, OPB-10, and OPR-01, allowed adequate discrimination between strains of A. parasiticus and A. sojae in RAPD analyses. A. sojae was further separated into group I and group II with the three primers. On the other hand, A. parasiticus was divided into group A and group B when amplified with OPA-04 and OPR-10 primers. The previously misidentified stain CCRC 32423 and the misclassified strain CCRC 30227 were identified as Aspergillus flavus and A. sojae, respectively, on the basis of RAPD patterns and morphological characteristics. We suggest that the RAPD technique is a rapid and reliable tool to distinguish A. parasiticus from A. sojae.