Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.     

Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.     

Three subunit proteins of membrane enzymes in mitochondria of Neurospora crassa contain a pantothenate derivative

Three proteins of the inner mitochondrial membrane of Neurospora crassa were found to be covalently modified with a derivative of pantothenic acid. One of these proteins is a subunit of cytochrome c oxidase and two are subunits of the ATPase-ATP synthase. Cells of a pantothenate auxotroph of N. crassa were labeled with [14C]pantothenic acid, and mitochondrial proteins containing radiolabeled pantothenate were detected by electrophoresis of detergent-solubilized mitochondria. Mitochondria from cells that were colabeled with [14C]pantothenate and [3H]leucine were reacted with specific antisera against the cytochrome c oxidase and F1-ATPase enzyme complexes. Electrophoresis of the labeled subunits of these isolated complexes showed that the [14C]pantothenate-associated peptides corresponded to [3H]leucine-labeled subunit 6 of cytochrome c oxidase and two [3H]leucine-labeled subunits (tentatively identified as subunits 8 and 11) of the ATPase-ATP synthase. Pantothenate modification of these enzyme subunits, which are synthesized on extramitochondrial ribosomes, may contribute to their transport and assembly into mitochondria, or it may participate in the catalytic activity of the assembled enzymes.     

Diploids in Microsporum gypseum

The paper studies diploids in dermatophyteMicrosporum gypseum. They were isolated as the more rapidly growing sectors from heterokaryons on minimal medium. They are characterized by their wild morphology, conidiation and growth rate, and they are prototrophic. In their genome they contain all the markers present in both mutant components.