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J Engberg

Publications and source records attributed to J Engberg.

At least 73 records · Page 4Linked to original sources
PubMed1988

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA.

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Amino Acid Sequence↗
PubMed1988

The secondary structure of large-subunit rRNA divergent domains, a marker for protist evolution.

The secondary structure of the large-subunit ribosomal RNA (24-26S rRNA) has been studied with emphasis on comparative analysis of the folding patterns of the divergent domains in the available protist sequences, that is Prorocentrum micans (dinoflagellate), Saccharomyces carlsbergensis (yeast), Tetrahymena thermophila (ciliate), Physarum polycephalum and Dictyostelium discoideum (slime moulds), Crithidia fasciculata and Giardia lamblia (parasitic flagellates). The folding for the D3, D7a and D10 divergent domains has been refined and a consensus model for the protist 24-26S rRNA structure is proposed. Two hundred seventy-seven nucleotide-long aligned sequences representing all or part of the D3, H32-33, D8, D9 and D10 divergent domains are used for the construction of unrooted phylogenetic trees either calculated from a nucleotide difference matrix, or determined with the PAUP programme based on the parsimony method. Both phylogenies suggest three major branchings, the first leading to the dinoflagellate (which branches off first), ciliate and yeast, the second to the slime moulds, and the last to the parasitic flagellates.

Animals↗
PubMed1987

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon.

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Animals↗
PubMed1986

Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups.

Previous work has demonstrated the presence of a self-splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups. In an attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree. This phylogeny was consistent with the groupings suggested by comparisons of other biochemical characters including cytoskeletal proteins, isozyme analyses, and restriction maps of complete rRNA transcription units. The homology groupings that were based upon the intron sequence data do not agree with the relationships inferred from the small subunit rRNA sequence data. These observations are taken to indicate that the intron character has been acquired independently in different species at a stage later than the branching out of the species.

Animals↗
PubMed1986

An intron in a ribosomal protein gene from Tetrahymena.

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.

Journal Article↗
PubMed1985

Sequence comparison of the rDNA introns from six different species of Tetrahymena.

We have studied the sequence variation of the rDNA intron among six species of Tetrahymena. From these data, the intron appears to be relatively well conserved in evolution. We have evaluated the sequence variations among the most distant of these species in relation to the secondary structure model for the intron RNA of Cech et al. (Proc. Natl. Acad. Sci. U.S.A. 80, 3903 (83)). Most of the sequence variation in the four new sequences reported here is found in single stranded loops in the model. However, in four cases we found nucleotide substitutions in duplex stem regions, two of them involving compensating base pair changes. Interestingly, one of these is found in a region that is known to be dispensable in the in vitro splicing reaction suggesting differences between the in vivo and in vitro reactions. One of the single nucleotide deletions is found in the so-called "internal guide sequence" which has been implicated in the alignment process during splicing. In conclusion, none of the observed natural sequence variations are in disfavor of the proposed secondary structure model.

Base Sequence↗
PubMed1985

Small nuclear RNAs in the ciliate Tetrahymena.

We have isolated and partially characterized a family of small nuclear RNAs (snRNAs) from three different species of the protozoan Tetrahymena. We find six distinct snRNAs ranging in size from 100 to 250 nucleotides. The two largest snRNAs, as well as an abundant, heterogenous group of smaller snRNAs are found in the nucleolar RNA fraction. None of the snRNAs are transcription products of the ribosomal RNA gene or its flanking regions, as shown by hybridization tests. The snRNAs are metabolically stable as determined by pulse/chase experiments and several of them contain a number of modified nuclotides. The snRNAs from Tetrahymena all have slightly different sizes from mammalian snRNAs. The cap structure of the snRNAs from Tetrahymena differs from that of the snRNAs from mammalian cells, but has not yet been fully characterized. The relative amount of snRNAs to total RNA is less in Tetrahymena (greater than 0.1%) than in mammalian cells (2%).

Animals↗
PubMed1985

Functional intron+ and intron- rDNA in the same macronucleus of the ciliate Tetrahymena pigmentosa.

Diallelic clones of Tetrahymena pigmentosa containing equal amounts of intron+ and intron- rDNA in the macronucleus were constructed. The macronucleus of the resulting strains divides amitotically during vegetative growth and the diallelic genotype is therefore unstable. The coexistence of the two alleles was followed in the total culture and in single cells during their vegetative segregation and it was observed that replication was non-preferential with respect to the two alleles. The diallelic clones were also used to demonstrate that intron-containing rDNA was transcribed and the transcript processed in the presence of corresponding intron- rDNA. The results are discussed in the light of the 'non-function' idea for ribosomal RNA introns.

Alleles↗
PubMed1985

Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species.

The extrachromosomal rDNA molecules from a number of Tetrahymena strains were characterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species.

Alleles↗
PubMed1985

The ribosomal RNA genes of Tetrahymena: structure and function.

This report reviews the structural and functional characteristics of the ribosomal RNA genes (rDNA) of the ciliate protozoan, Tetrahymena. The study of rDNA was initiated some 10 years ago in order to establish a model system for rRNA gene expression in lower eukaryotes. The system proved very useful for studying several aspects of rRNA gene expression so that a considerable amount of information on rDNA structure and function has accumulated during the past years as a result of studies done in several laboratories. The initial finding that starved Tetrahymena cells, upon refeeding, preferentially replicated their rDNA for up to 90 min before bulk macronuclear DNA synthesis resumed, greatly facilitated the first studies. This observation permitted the rDNA to be labeled selectively with isotopes and in this way it was possible to isolate rDNA in pure form, to study replicative intermediates of the rDNA and to study rDNA containing chromatin in intact nuclei without resorting to chromatin fractionation. As a result of these studies it was found that the macronuclear rDNA of all Tetrahymena species consisted of a homogeneous population of extrachromosomal, linear molecules with a size of about 20 kilobase-pairs (kb) containing two identical transcription units for pre-rRNA arranged in a reverse repeat orientation (palindromic symmetry). The origins of replication were localized to the central non-transcribed spacer region and it was shown that this region has a chromatin structure which is different from that of the transcribed region. The primary DNA sequence is now known in many parts of the rDNA molecule, including the central part (containing the replication origins), the telomeric parts and the regions containing the sites of transcription initiation and termination. Transcription studies demonstrated the presence of an intervening sequence (intron) in the 26S rRNA coding region in some strains of Tetrahymena. Interestingly, the intron is transcribed and later removed from the primary transcript as a result of a rather unusual reaction which can take place in vitro in the absence of added protein factors. The finding of interbreeding strains of the intron+ and intron- rDNA genotype provided physical markers on the genes and have made possible a description of the inheritance and allelic assortment of the Tetrahymena rDNA. These studies proved that the free, palindromic rDNA molecules of the macronucleus arise from a chromosomally integrated, micronuclear rDNA copy as a result of a conjugational dependent amplification event, and that the intron is inherited as a neutral character during sexual and vegetative reproduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1984

Nucleotide sequence of the 5'-terminal coding region for pre-rRNA and mature 17S rRNA in Tetrahymena thermophila rDNA.

The 5'-terminus of 35S pre-rRNA and mature 17S rRNA of Tetrahymena thermophila was mapped on cloned rDNA fragments by S1 nuclease protection experiments. A single site for transcription initiation was observed when pre-rRNA prepared by three different methods was used as RNA probe. These mapping results were unambiguously confirmed by sequencing the 5'-terminal region of in vitro capped 35S pre-rRNA. DNA sequence analysis of about 520 nucleotides upstream of the transcription initiation site revealed several distinct sets of highly conserved repeat sequences. In addition, the 840 nucleotides downstream of the transcription initiation site (+ 1) was determined and shown to include the 5'-terminus of the 17S rRNA coding region at position + 647. A region surrounding the position + 195 contains an inverted repeat sequence which could be the structural basis for the recently described premature transcription termination event in this organism (Kister et al. (1983) Nucl. Acids Res. 11, 3487-3502).

Animals↗
PubMed1983

Strong sequence conservation of a 38 bp region near the center of the extrachromosomal rDNA palindrome in different Tetrahymena species.

The restriction-endonuclease map and the nucleotide sequence of the central region in the extrachromosomal rDNA palindrome of two micronuclear and one a-micronucleate species of Tetrahymena has been determined. The sequence data show that the different species investigated have a 24 or 26 nucleotide sequence region at the very center of the rDNA molecule which is non-palindromic. Comparison of the present sequence data with the published data of another micronucleate species reveal that a segment of 38 base pairs just outside the non-palindromic center is highly conserved in all the different species, while the rest of the central region show little sequence homology. The relevance of this conserved region to the amplification process of the rDNA molecule is discussed.

Animals↗
PubMed1982

The nucleotide sequence at the transcription termination site of the ribosomal RNA gene in Tetrahymena thermophila.

The sequence of 415 nucleotides surrounding the transcription termination site for ribosomal RNA in Tetrahymena thermophila has been determined. The positions of the 3'-ends of mature 26S rRNA, pre-26S rRNA and 35S pre-rRNA were localized within this sequence by hybridization of the purified RNA species to be selected DNA fragments, followed by S1 nuclease treatment of the hybrid and a precise sizing of the RNA-protected DNA fragments on sequencing gels. The 35S pre-rRNA population contained molecules with two distinct 3'-ends, one of which is identical to the end of pre-26S and 26S rRNA, while the other corresponds to a position 15 nucleotides further downstream, which is assumed to be the transcription termination site. The non-coding DNA strand contains a cluster of T's at the putative termination site, and several other T clusters are found further downstream. A short inverted repeat sequence is located near the putative termination site within the transcribed region. The possible role of these structures for transcription termination is discussed.

Animals↗
PubMed1979

The intervening sequence in the 26S rRNA coding region of T. thermophila is transcribed within the largest stable precursor for rRNA.

We studied the transcription of the intervening sequence in the 26S rRNA coding region of the extrachromosomal rDNA molecules in the macronucleus of T. thermophila by hybridization of purified nuclear rRNA precursors or cytoplasmic 26S rRNA to purified native rDNA or specific rDNA restriction fragments. Examination of R loop hybrids in the electron microscope and analyses of S1-protected rDNA fragments in alkaline agarose gels showed that mature 26S rRNA, nuclear pre-26S rRNA and a fraction of the pre-rRNA molecules containing both the sequences for 17S and 26S rRNA all lack the region corresponding to the intervening sequence. The rest of the pre-rRNA molecules, however, hybridize in a colinear fashion to the whole coding region, and thus must contain the intervening sequence. We can conclude from these results that the intervening sequence is transcribed within the primary transcription product of the rDNA, and that the post-transcriptional removal of the intervening RNA sequence is a very early processing event in the organism.

Animals↗