Fernanda Appleton
Biology 1615
Research Paper:” The Oxytricha trifallax Macronuclear Genome: A Complex
Eukaryotic Genome with 16,000 Tiny Chromosomes”
Introduction:
The macronuclear genome of the ciliate Oxytricha trifallax, contained in its
somatic nucleus, has a unique genome architecture. Unlike its diploid germline genome,
which is transcriptionally inactive during normal cellular growth, the macronuclear
genome is fragmented into at least 16,000 tiny (~3.2 kb mean length) chromosomes, most
of which encode single actively transcribed genes and are differentially amplified to a
few thousand copies each. The smallest chromosome is just 469 bp, while the largest is
66 kb and encodes a single enormous protein. They found considerable variation in the
genome, including frequent alternative fragmentation patterns, generating chromosome
isoforms with shared sequence, also has a limited variation in chromosome amplification
levels, though insufficient to explain mRNA transcript level variation. Two fundamental
differences distinguish Oxytricha's macronuclear chromosomes from those of
Tetrahymena and Paramecium: Oxytricha's chromosomes are tiny
(“nanochromosomes,” with a mean length ~3.2 kb reported in this study), each typically
encoding just a single gene with a minimal amount of surrounding non-protein-coding
DNA.
Reason for Research:
In this Research Paper, the Scientists were searching information about the
macronuclear genome of the ciliate Oxytricha trifallax, displays extreme and unique
eukaryotic genome architecture with extensive genomic variation. They compare
Oxytricha trifallax, Tetrahymena thermophila, and Paramecium tetraurelia in this study.
Oxytricha trifallax is a distinctive ciliate, an ancient lineage of protists named for
their coats of cilia. Like all ciliates, Oxytricha has two types of nuclei: a
micronucleus, a germline nucleus that is largely transcriptionally inactive during
vegetative growth, and a macronucleus, which is the transcriptionally active somatic
nucleus.
Material and Methods:
In laboratory culture conditions, Oxytricha trifallax tends to replicate asexually
and rarely conjugates the result is meiotic recombination. Conjugation in the
laboratory is induced by starvation as long as cells of compatible mating types are
available, but the Scientists do not know if this also can occur in a natural
environment. Oxytricha trifallax strain JRB310 was cultured in inorganic salts
medium and starved for 3 d at 4°C to allow consumption of most of the food source
(Chlamydomonas reinhardtii) in culture. Cells were harvested by filtering through
several layers of gauze to remove large particles. Then, a 10 µm Nitex membrane was
used to concentrate cells and remove small contaminants.
Results:
They predict that Oxytricha nanochromosomes (80%) contain single genes.
They use many techniques, one mentioned in the article was to obtain pure macronuclear
DNA for Illumina sequencing, Oxytricha trifallax strain JRB310 was cultured in
inorganic salts medium and starved for 3 d at 4°C to allow consumption of most of the
food source (Chlamydomonas reinhardtii) in culture.
The macronuclear genome encodes all the genes required for vegetative
growth, consistent with the observation of amicronucleate Oxytricha species capable of
vigorous growth in laboratory conditions.
The macronuclear genome inherits considerable diversity from the
micronuclear genome, in the form of abundant nucleotide polymorphisms (SNP
heterozygosity is ~4.0%). Additional macronuclear genome variation arises during and
after nuclear development, through a complex interplay between genetic and epigenetic
forces, resulting in modest variation in DNA amplification levels and TASs, as well as
thousands of alternative nanochromosome isoforms.
Conclusion:
The Scientist found considerable variation in the genome, including frequent
alternative fragmentation patterns, generating chromosome isoforms with shared
sequence; they also discovered limited variation in chromosome amplification levels,
though insufficient to explain mRNA transcript level variation.