Identification of a specific telomere terminal transferase activity in tetrahymena extracts

The telomerase enzyme of Tetrahymena synthesizes repeats of the telomeric DNA sequence TTGGGG de novo in the absence of added template. The essential RNA component of this ribonucleoprotein enzyme has now been cloned and found to contain the sequence CAACCCCAA, which seems to be the template for the synthesis of TTGGGG repeats.

A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.

https://www.cell.com/cell/pdf/0092-8674(89)90035-4.pdf

The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats

Microorganisms have been mutating and evolving on Earth for billions of years. Now, a field of research has developed around the idea of using microorganisms to study evolution in action. Controlled and replicated experiments are using viruses, bacteria and yeast to investigate how their genomes and phenotypic properties evolve over hundreds and even thousands of generations. Here, we examine the dynamics of evolutionary adaptation, the genetic bases of adaptation, tradeoffs and the environmental specificity of adaptation, the origin and evolutionary consequences of mutators, and the process of drift decay in very small populations.

http://personales.upv.es/sfelena/Nature%20Rev.%20Genetics%201.pdf

Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation.

The telomere terminal transferase of tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity

Strains of the bacterium Escherichia coli habouring genes that increase mutation rates are known to have an evolutionary advantage in chemostat competition over otherwise isogeneic strains with lower mutation rates1–3. This advantage is frequency-dependent, the mutator strain being favoured only above a starting ratio of ∼5×10−5, and it results from the fact that the necessary beneficial mutations cannot be generated in a mutator population below a certain size3. Here we consider the possibility that the mutagenic4,5 properties of transposable elements confer an advantage in the same manner as mutator genes. A previous report has shown that the transposon Tn5 increases the fitness of E. coli in chemostats, although the reason for this effect has not been established6. Our results show that the transposon Tn10 also confers an advantage in chemostats. In addition, we find that (1) this advantage, like that associated with mutator genes, is frequency-dependent, (2) whenever the Tn10 strains win, a segment of Tn10, probably its IS10 sequences, has undergone transposition to a new site, (3) the new insertions converge into a site contained within a 3.2 kilobase (kb) PvuII fragment of the genome, and (4) no transpositions are detected when the Tn10 population lses. We conclude that Tn10 confers an advantage by increasing the mutation rate of the host bacterium.

Transposable elements as mutator genes in evolution

The saturation values for ribosomal RNA hybridization have been determined for DNA from diploid and polytene tissues of Drosophila melanogaster. These values have been measured in XO and XX larvae which have, respectively, one and two nucleolus organizers in the diploid chromosome set. The results show that (1) in diploid cells the ribosomal (r)DNA is present in amounts proportional to the number of nucleolus organizers, (2) in polytene cells the rDNA is under-replicated with respect to the euchromatic DNA, and (3) in polytene cells the amount of rDNA is independent of the diploid number of nucleolus organizers. These observations suggest that somatic variations in rDNA content may involve independent polytenization of the nucleolus organizer without change in the number of ribosomal cistrons per organizer. The independent polytenization of rDNA is proposed as the chromosomal explanation for the relative increase in rDNA in flies of the XO constitution.

https://www.pnas.org/content/pnas/70/5/1359.full.pdf

Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster.

The termini of macronuclear DNA molecules from the protozoan Oxytricha fallax share a common sequence and structure, both of which differ markedly from those deduced for yeast telomeres. Despite these differences, terminal restriction fragments from O. fallax macronuclear DNA can support telomere formation in yeasts. Two linear plasmids (LYX-1 and LYX-2) constructed by ligating BamHI-digested total Oxytricha macronuclear DNA to a yeast vector were analyzed. One end of LYX-1 and both ends of LYX-2 are derived from the Oxytricha DNA that encodes rRNA (rDNA) whereas the other end of LYX-1 is from an Oxytricha fragment other than rDNA. After propagation in yeast, both ends of LYX-1 and LYX-2 retain the C4A4 repeat characteristic of the O. fallax terminal sequence. In addition, both ends of both plasmids acquire 300-1000 base pairs of DNA containing the sequence (C-A)n, a sequence found near the termini of yeast chromosomes. Thus, at least two different Oxytricha termini display distinctive properties in yeast cells in that linear plasmids containing them are not degraded nor are they integrated into chromosomal DNA. These Oxytricha termini may act directly as telomeres in yeast; alternatively, the Oxytricha DNA may serve as a signal that results in the elaboration of a yeast telomere on the ciliate DNA.

https://www.pnas.org/content/pnas/81/5/1475.full.pdf

Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA.

The hypotrichous ciliated protozoan Oxytricha fallax possesses a mechanism for processing its genome into a collection of small, gene-sized, transcriptionally active sections. Macronuclear DNA is arranged as short achromosomal sections, 0.5–22 kilo-base pairs (kbp) long, while micronuclear DNA has a typical chromosomal organization1,2. Macronuclear DNA is derived from micronuclear DNA by a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity3,4. The isolation of intact macronuclear DNA sections enables the study of specific structural units of genetic material that occur naturally in the organism. We have previously described5 the construction of a recombinant plasmid, pOfACT (1.6), which contains an intact 1.6-kb length of macronuclear DNA (Fig. 1) homologous to a yeast actin gene6. We now present the complete nucleotide sequence of the cloned DNA molecule and the derived sequence of the actin protein which it encodes. As the sequence is that of a complete, naturally occurring DNA molecule, it shows all cis-active regulatory regions in addition to the coding region. The actin protein encoded differs considerably from other known actins.

Nucleotide sequence of a macronuclear gene for actin in Oxytricha fallax.

DNA in the macronuclei of Oxytricha fallax, as in other hypotrichous ciliate protozoa, exists as small, achromosomal molecules rather than in chromosomes. We report studies on O. fallax DNA using physicochemical procedures and nucleic acid hybridization. Macronuclear DNA molecules range in size from 22 kilobase pairs (kb) to about 0.5 kb. The DNA has a buoyant density in CsCl of 1.694 g·cm-3 and a melting temperature in 15 mM NaCl/1.5 mM sodium citrate, pH 7, at 65.4°. These values correspond to 34.7% Gua + Cyt and 28.1% Gua + Cyt, respectively, and base composition determined by thin-layer chromatography of nucleotides is 32.4% Gua + Cyt. The only modified nucleotide that is detectable is N6-methyldeoxyadenylate (0.2%), and the amount and kind of modification cannot account for the discrepancies in nucleotide composition determination by the three methods. The genes for 25S and 19S rRNA are contained in DNA molecules 6.67 kb in length, of which at least 6.15 kb is transcribed. These rDNA molecules show no intrastrand complementarity as does rDNA in some other lower eukaryotes, and they have two asymmetric sites recognized by endonuclease EcoRI. The genes for 5S RNA are in DNA molecules 0.69 kb in length. Digestion of this DNA with restriction enzymes BamHI, BsuI, HhaI, and TaqI gives no evidence for a tandemly repeated sequence. It is likely that both 19S + 25S rRNA genes and 5S RNA genes in the Oxytricha macronucleus exist as single transcription units, and both may have “spacer” regions approximately 0.5 kb long.

http://www.pnas.org/content/75/10/4992.full.pdf

Brian B. Spear

Papers

Transposable elements as mutator genes in evolution

Independent control of ribosomal gene replication in polytene chromosomes of Drosophila melanogaster.

Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA.

Nucleotide sequence of a macronuclear gene for actin in Oxytricha fallax.

Macronuclear DNA of the Hypotrichous Ciliate Oxytricha fallax