Characterization of two abundantly expressed constitutive genes of Neurospora crassa

Two abundantly expressed, constitutive genes of Neurospora crassa were isolated during differential screening of Neurospora genomic libraries. The coding regions of these two genes, designated RLF1 and RLF3, were identified by hybridization of the cloned DNA sequences with cDNA probes made from polyadenylated RNA. The RLF3 gene was carried on a 15-kilobase Neurospora BamHI DNA fragment present in a lambda 1059 recombinant; a 2-kilobase restriction fragment that contains RLF3 was subcloned into plasmid pBR322 prior to further characterization. Southern blot analysis revealed that both RLF1 and RLF3 are single copy genes. Northern blot analysis and S1 nuclease mapping demonstrated that RLF1 is transcribed to yield a 1.6-kilobase RNA, whereas RLF3 appears to give rise to two distinct sized RNA species of 1.0 and 1.6 kilobases. RNA dot blot analysis provided conclusive evidence that both of these genes are constitutively expressed. These constitutive genes will be valuable to provide a detailed comparison with the 5' flanking regions of regulated genes.     

Nucleotide sequences of the 5S ribosomal RNA genes and their adjacent regions in Schizosaccharomyces pombe.

The organization of 5S ribosomal RNA (rRNA) genes in the genome of Schizosaccharomyces pombe has been investigated by restriction and hybridization analyses. The 5S rRNA genes were not linked to the other three species of rRNA genes which formed a repeating unit of 6.9 megadaltons, but located in other regions surrounded by heterogeneous sequences. The 5S rRNA gene organization in S. pombe is therefore different from those in other yeasts; Saccharomyces cerevisiae and Torulopsis utilis. Four restriction segments of different sizes each containing a single 5S rRNA gene were cloned on a bacterial plasmid, and the sequences in and around the RNA coding regions were determined. In the RNA coding regions, the sequences in four clones were identical with an exception that one residue has been substituted in one clone. In the flanking regions, the sequences were extremely rich in the AT-content and highly heterogeneous. The sequences were also markedly different from those in the corresponding regions of the other two yeasts. THe presence of T-clusters in the regions immediately after the RNA coding sequences was only notable homology among the four clones and the other two yeasts.     

Schistosoma haematobium: analysis of eggshell protein genes and their expression

Eggshell protein genes of Schistosoma mansoni that encode a 14 kDa protein have been shown to be highly conserved and expressed in a sex-, tissue-, and temporal-specific manner. To initiate studies on the eggshell protein genes of S. haematobium, a cDNA probe, pSMf 61-46, representing a S. mansoni eggshell protein mRNA was used to screen a S. haematobium genomic library. Of the seven independent recombinant clones isolated, two (lambda SH 2-1 and lambda SH 6-1) were analyzed and compared to those of S. mansoni. lambda SH 2-1 and lambda SH 6-1 each contain a different genomic copy of the gene encoding a 19.8 and 17.6 kDa protein, respectively. This is due to an additional 78 bp present in the coding region of lambda SH 2-1 relative to lambda SH 6-1. The rest of the coding sequences are identical, and the 5' and 3' untranslated regions are nearly identical. The deduced amino acid sequences of S. haematobium eggshell proteins are very rich in glycine (47 and 50%) when compared to 43.5% glycine in the protein encoded by S. mansoni. Long stretches of glycines, as many as 15 in a row, occur in the S. haematobium sequence. DNA comparison of the eggshell protein genes of the two schistosome species yielded an overall homology of 83.1%. The homology is much higher in the 5' and 3' untranslated regions than in the protein-coding regions. Genomic clones of both species contained second open reading frames, which appeared to be kept open as a consequence of the amino acid composition of the other. There are no introns in S. haematobium or S. mansoni eggshell protein genes, and the genomic Southern data indicated a similar arrangement of these genes in the genome of both species. Primer extension experiments and dideoxynucleotide sequencing of the RNA determined the mRNA cap site sequence as ATCAT and ATCAC in lambda SH 2-1 and lambda SH 6-1, respectively. Northern blot analysis determined the size of the mRNA to be about 1.0 kp. Expression of the RNA from these genes appears to be regulated in a manner similar to the corresponding genes in S. mansoni. mRNA is found only in mature females and first appears at 70 days after infection of hamsters. DNA sequence comparisons of the 5' flanking regions of S. haematobium and S. mansoni eggshell protein genes to each other and to those of silkmoth and Drosophila revealed several short sequence elements that are shared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structures of the genes encoding the alpha and beta subunits of the Neurospora crassa mitochondrial ATP synthase.

We have isolated and sequenced cDNA and genomic clones encoding the alpha and beta subunits of the Neurospora crassa ATP synthase. The genes are not linked to each other: atp-1(alpha) maps to either linkage group I or V, and atp-2(beta) lies on linkage group II. The two genes resemble each other in having a large number of introns, five in atp-1 and seven in atp-2, mostly positioned near their 5' ends and varying in length from 60-332 bp. The coding regions of both genes have a high G+C content (59%) and use a low number of codons, 46 (atp-1) and 44 (atp-2), a feature associated with highly expressed genes. Northern-blot analysis shows both genes are expressed at high levels during mycelial growth. Comparison of the exon-intron structures of the beta-subunit-encoding gene with those from human and tobacco showed a similar number of introns, several closely positioned, but no exact conservation in position, size or sequence of introns.     

Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors

Active minichromosomes assembled on injected 5S RNA gene clones are stable in Xenopus oocytes; endogenous 5S DNA specific factor(s) are required for their assembly. When somatic-type and oocyte-type 5S RNA gene clones are coinjected, the somatic genes are assembled into active minichromosomes, while most of the oocyte genes are assembled into inactive ones. The differential 5S RNA gene expression, which mimics that in somatic cells, appears to result from titration of 5S DNA specific factor(s) by the competing somatic 5S DNA, followed by histone mediated assembly of inactive chromatin on the oocyte 5S DNA. Stable minichromosomes are also assembled on a cloned histone H4 gene; again, intragenic DNA rearrangements affect the efficiency of assembly of active chromatin and differential gene expression occurs after coinjection of two or more H4 DNA constructs. We suggest that the H4 DNA molecules also compete for limiting quantities of specific DNA binding factor(s) required for the assembly of active H4 gene chromatin.     

Linkage of 5S RNA and 16S+23S RNA genes on the E. coli chromosome.

Summary Episomes carrying limited regions of the chromosome where 5S RNA genes have previously been located are described. The DNA purified from each of these episomes contains one gene per molecule for each of the three ribosomal RNA species as shown by hybridization experiments.This work was supported in part by a grant from the C.E.A.     

Ribosomal Gene Structure, Variation and Inheritance in Maize and Its Ancestors

We have examined the structure of nuclear genes coding for ribosomal RNAs in maize and its wild relatives, the teosintes and Tripsacum. Digestion of the rDNA (genes coding for 18S, 5.8S and 26S RNAs) with 15 restriction endonucleases (with six base pair recognition sites) yields essentially a single map for the approximately 10,000 repeat units within an individual plant or species. Both length and site variation were detected among species and were concentrated in the intergenic spacer region of the rDNA repeat unit. This result is in agreement with patterns of rDNA change observed among wheat and its relatives (Triticeae), and among vertebrate species. Digestion of these nuclear DNAs with BamHI and subsequent hybridization with a 5S RNA gene-specific probe allowed determination of the size of the 5S gene repeat unit in maize, teosintes, and Tripsacum. Groupings in the genus Zea were characterized by distinct repeat unit types five Tripsacum species examined shared a 260 base pair major repeat unit type. Additionally, several other restriction endonuclease cleavage patterns differentiated among the 5S DNAs within the genus Zea. The rDNA and 5S DNA restriction site variation among the species can be interpreted phylogenetically and agrees with biochemical, karyotypic, and morphological evidence that places maize closest to the Mexican teosintes. For both gene arrays, contributions from each parental genome can be detected by restriction enzyme analysis of progeny from crosses between maize and two distantly related teosintes, Zea luxurians or Zea diploperennis, but certain teosinte arrays were underrepresented in some of the hybrids.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

The 5.8S RNA gene sequence and the ribosomal repeat of Schizosaccharomyces pombe

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.     

Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.