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Biomedical subjects

B C Lu

Publications and source records attributed to B C Lu.

At least 19 recordsLinked to original sources
PubMed2000

The control of meiosis progression in the fungus Coprinus cinereus by light/dark cycles.

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.

Circadian Rhythm↗
PubMed1997

Fungal galectins, sequence and specificity of two isolectins from Coprinus cinereus.

Galectins are members of a genetically related family of beta-galactoside-binding lectins. At least eight distinct mammalian galectins have been identified. More distantly related, but still conserving amino acid residues critical for carbohydrate-binding, are galectins in chicken, eel, frog, nematode, and sponge. Here we report that galectins are also expressed in a species of fungus, the inky cap mushroom, Coprinus cinereus. Two dimeric galectins are expressed during fruiting body formation which are 83% identical to each other in amino acid sequence and conserve all key residues shared by members of the galectin family. Unlike most galectins, these have no N-terminal post-translational modification and no cysteine residues. We expressed one of these as a recombinant protein and studied its carbohydrate-binding specificity using a novel nonradioactive assay. Binding specificity has been well studied for a number of other galectins, and like many of these, the recombinant C. cinereus galectin shows particular affinity for blood group A structures. These results demonstrate not only that the galectin gene family is evolutionarily much older than previously realized but also that fine specificity for complex saccharide structures has been conserved. Such conservation implies that galectins evolved to perform very basic cellular functions, presumably by interaction with glycoconjugates bearing complex lactoside carbohydrates resembling blood group A.

Amino Acid Sequence↗
PubMed1993

Spreading the synaptonemal complex of Neurospora crassa.

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.

Genome, Fungal↗
PubMed1992

Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding gene from the basidiomycete Coprinus cinereus.

The naturally synchronous meiosis of the fungus, Coprinus cinereus, provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a lambda gt11 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene (MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.

Amino Acid Sequence↗
PubMed1991

An endo-exonuclease from meiotic tissues of the basidiomycete Coprinus cinereus. Its purification and characterization.

An endo-exonuclease has been identified and partially purified from the basidiocarp tissues of the basidiomycete Coprinus cinereus, which include synchronous meiosis at karyogamy-pachytene stages. Its peak activity appears during the meiotic prophase. The Coprinus endo-exonuclease has a single-strand specific endonuclease activity that converts the supercoiled DNA to relaxed DNA. The endonucleolytic cleavage of single-strand DNA generates 3'-phosphomonoester termini. It is also a single-strand-specific exonuclease and it hydrolyzes linear DNA in a 3' to 5' direction, but is unable to hydrolyze single-strand DNA having a 3'-phosphomonoester terminus. It requires Mg2+ with an optimal concentration of 25 mM. It has an optimal pH of 8.3, a peak enzyme activity at 50 degrees C, and it contains a single 43-kilodalton polypeptide. Coprinus meiotic endo-exonuclease may be involved in the substrate preparation for meiotic recombination.

Basidiomycota↗
PubMed1990

Involvement of Coprinus endonuclease in preparing substrate for in vitro recombination.

A functional recombination assay involving the tetracycline mutant plasmids, pUW1 and pUW4, was used to assess (i) the nature of the DNA substrates needed and (ii) the involvement of Coprinus endonuclease in preparing substrate, for the RecA-directed recombination process. A gapped circular plasmid and a linear or a nicked circular plasmid are efficient substrate combinations in this system to achieve a 160-fold increase in the in vitro recombination frequency over the control levels. The Coprinus endonuclease obtained from early meiotic prophase can produce such substrates. The recombination frequency obtained with the combination of gapped pUW1 plasmids initially relaxed by the Coprinus endonuclease and linear pUW4 plasmids produced by the site-specific BamHI digest is 10-fold lower than that obtained when both substrates are digested by BamHI. The results suggest that the Coprinus endonuclease creates random nicks on plasmid DNA. Glyoxal gel electrophoretic analysis was used to confirm this random nicking activity of Coprinus endonuclease.

Agaricales↗
PubMed1988

Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus.

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.

Agaricales↗
PubMed1985

Silver staining of meiotic chromosomes in the fungus, Coprinus cinereus.

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.

Basidiomycota↗
PubMed1984

The cellular program for the formation and dissolution of the synaptonemal complex in Coprinus.

Inhibition of protein synthesis by cycloheximide on processes in meiosis was used to probe the cellular program for the formation and dissolution of the synaptonemal complex (SC) in the synchronous meiotic system of Coprinus cinereus. The pathway for the synthesis and assembly of the synaptonemal complex is proposed to be as follows: (1) synthesis and assembly of lateral components on the chromosomes; (2) synthesis and assembly of the central components in the nucleolus; (3) the lateral components of the homologous chromosomes are brought together to pair when the homologous pairing occurs at zygotene; (4) the transport of the central components from the nucleolus to join the paired lateral components and thus complete the synaptonemal complex. Continued protein synthesis is required for all steps. Step (1) is nearly complete 2 h after the onset of karyogamy, because continued assembly is possible in the presence of cycloheximide. The transition point for step (2) is 4 h after the onset of karyogamy, as inhibition at this point results in accumulation of central components in the nucleolar dense body. The paired lateral components of step (3) are deprived of the central component. The transition point for step (4) is 5 h after the onset of karyogamy, for inhibition at this point no longer prevents transport. Two steps are proposed for dissociation and dissolution of the SC at the end of pachytene. Protein synthesis is required for the dissolution of SC. Inhibition at this point causes accumulation of polycomplexes. Mutations in various organisms from the literature relating to the SC support the validity of the proposed pathway.

Agaricales↗
PubMed1982

Meiosis in Coprinus: characterization and activities of two forms of DNA polymerase during meiotic stages.

Two forms of DNA polymerase have been studied in the basidiomycete Coprinus. DNA polymerase from basidiocarp tissues at zygotene-pachytene stage has been purified 3,500-fold and defined as DNA polymerase b by virtue of its insensitivity to N-ethylmaleimide and by its low molecular weight (76,000). This enzyme has optimal activity at pH 7.0 to 7.5, at 200 mM KCl, and at 25 degrees C incubation temperature. It can use polycytidylic acid-oligo(dG)12-18 as template primer in addition to homodeoxypolymers. The DNA polymerase a is mainly produced in the exponentially growing mycelium. It is sensitive to N-ethylmaleimide and has a temperature optimum at 35 degrees C. At the premeiotic S phase, activities from both polymerase a and polymerase b are found in cell-free extracts. The b enzyme is the only DNA polymerase produced during meiotic prophase. Its assayable activity exhibits two peaks, one at premeiotic S stage and one at pachytene. It is possible that DNA polymerase b is responsible for pachytene repairs involved in recombination.

DNA-Directed DNA Polymerase↗
PubMed1980

The effect of hydroxyurea on meiosis and genetic recombination in the fungus Coprinus lagopus.

Treatment with hydroxyurea (HU) inhibits fruitbody development in Coprinus lagopus (sensu Lewis). The two most sensitive meiotic stages are mid-late premeiotic S phase and the pachytene-diplotene period. A 2 or 4 h treatment at mid-late premeiotic S phase arrests fruitbody development. If the same treatment is given at pachytene and diplotene, the fruitbody completes meiosis but the spores produced are inviable. The spores from fruitbodies treated at pachytene appear to be normal whereas those treated at diplotene are empty because the four nuclei remain in the basidium. A 2 or 4 h treatment with HU during karyogamy causes a decrease in spore viability and a dramatic increase in recombination frequency. The same treatment at metaphase II or later stages causes little damage to fruitbody development or spore viability. The effects of HU on the meiotic cell cycle suggest that the drug exerts its effect by inhibiting the synthesis of deoxynucleotides.

Agaricales↗
PubMed1978

Meiosis in Coprinus. VIII. A time-course study of the fusion and division of the spindle pole body during meiosis.

The time-course study of meiosis in the fungus Coprinus cinereus (C. lagopus) by electron microscopy reveals that two monoglobular spindle pole bodies (SPB's) of prekaryogamy nuclei come together during karyogamy and are fused. The fusion SPB of postkaryogamy nucleus persists through zygotene and pachytene as evidenced by the presence of axial components and synaptonemal complexes. At early diplotene, the SPB divides. The divided SPB takes on a diglobular form, which grows in size to form two daughter SPB's. These separate and move to opposite poles at metaphase I.

Agaricales↗
PubMed1976

Genetic recombination of Coprinus. V. Repair synthesis of deoxyribonucleic acid and its relation to meiotic recombination.

Repair synthesis of DNA per se at pachytene is not needed for commitment to meiotic recombination although it is a necessary event to follow. Recombination frequency is governed by the rate of nicking and the time in which unrepaired nicks are allowed to match and crossover. Cold treatment at pachytene prevented repair synthesis, hence open nicks were accumulated to match and crossover, and a 3-fold increase in recombination resulted. The kinetics of cold temperature effect followed a quadratic function as shown by a computer simulation which agreed with our experimental data (Lu, 1974b). High temperature did not change the rate of repair synthesis. It did cause an increased nicking which led to a twofold increase in recombination and which entailed a higher rate of recovery repair synthesis.

Agaricales↗
PubMed1975

Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.

Agaricales↗