Andre R. O. Cavalcanti

The Pathway to Detangle a Scrambled Gene

Background Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20–30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development. Methodology/Principal Findings To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways. Conclusions/Significance This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

MDS_IES_DB: a database of macronuclear and micronuclear genes in spirotrichous ciliates

Ciliated protozoa have two kinds of nuclei: Macronuclei (MAC) and Micronuclei (MIC). In some ciliate classes, such as spirotrichs, most genes undergo several layers of DNA rearrangement during macronuclear development. Because of such processes, these organisms provide ideal systems for studying mechanisms of recombination and gene rearrangement. Here, we describe a database that contains all spirotrich genes for which both MAC and MIC versions are sequenced, with consistent annotation and easy access to all the features. An interface to query the database is available at http://oxytricha.princeton.edu/dimorphism/database.htm.

Coding properties of Oxytricha trifallax (Sterkiella histriomuscorum) macronuclear chromosomes: analysis of a pilot genome project

The macronuclear genomes of spirotrichous ciliates are almost entirely polyploid, single-gene chromosomes (“nanochromosomes”). We recently performed a pilot genome project for a member of this group, Oxytricha trifallax (Sterkiella histriomuscorum), in which ∼2000 nanochromosomes were cloned at random and end-sequenced. Here we describe the global properties of the coding regions predicted for these molecules, including nucleotide composition, codon usage, and intron properties. In identifying splice donor, acceptor and branch sites, we found that longer introns in Oxytricha have a stronger signal at the donor site than do smaller introns, as has been found for Caenorhabditis elegans and Drosophila, despite the overall small size of the introns. A systematic search for multi-gene chromosomes identified 11 candidate nanochromosomes. We compare the results from this large dataset with those obtained from earlier studies and with statistics recorded from ciliates and other eukaryotes.

Decoding the decoding region: Analysis of eukaryotic release factor (eRF1) stop codon-binding residues

Peptide synthesis in eukaryotes terminates when eukaryotic release factor 1 (eRF1) binds to an mRNA stop codon and occupies the ribosomal A site. Domain 1 of the eRF1 protein has been implicated in stop codon recognition in a number of experimental studies. In order to further pinpoint the residues of this protein involved in stop codon recognition, we sequenced and compared eRF1 genes from a variety of ciliated protozoan species. We then performed a series of computational analyses to evaluate the conservation, accessibility, and structural environment of each amino acid located in domain 1. With this new dataset and methodology, we were able to identify eight specific amino acid sites important for stop codon recognition and also to propose a set of cooperative paired substitutions that may underlie stop codon reassignment. Our results are more consistent with current experimental data than previously described models.

Sequence Features of Oxytricha trifallax (Class Spirotrichea) Macronuclear Telomeric and Subtelomeric Sequences

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear nanochromosomes of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3 and 5 untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.

Conservation of tandem stop codons in yeasts

BackgroundIt has been long thought that the stop codon in a gene is followed by another stop codon that acts as a backup if the real one is read through by a near-cognate tRNA. The existence of such tandem stop codons, however, remains elusive.ResultsHere we show that a statistical excess of stop codons has evolved at the third codon downstream of the real stop codon UAA in yeasts. Comparative analysis indicates that stop codons at this location are considerably more conserved than sense codons, suggesting that these tandem stop codons are maintained by selection. We evaluated the influence of expression levels of genes and other biological factors on the distribution of tandem stop codons. Our results suggest that expression level is an important factor influencing the presence of tandem stop codons.ConclusionOur study demonstrates the existence of tandem stop codons, which represent one of many meaningful genomic features that are driven by relatively weak selective forces.

Interconversion of Germline-Limited and Somatic DNA in a Scrambled Gene

Ciliates have a somatic and a germline nucleus; after sexual conjugation a new somatic nucleus forms from the new zygotic germline nucleus. Formation of the somatic nucleus involves precise elimination of a large portion of DNA sequences from the germline. Here we compare the architecture of the germline and somatic versions of the actin I gene in two geographically isolated strains of Stylonychia lemnae. We show that the structure of the germline gene is surprisingly mercurial, with the distinction between germline-limited and somatic sequences variable over the course of evolution. This is, to our knowledge, the first example of evolutionary swapping of retained versus deleted sequences during ciliate development, with sequences deleted during development that are specifically retained in another strain.

VESTIGES OF EARLY MOLECULAR PROCESSES LEADING TO THE GENETIC CODE

We compare predictions from a proposed model for the origin of the genetic code (J. Theor. Biol (1993) 164, 291–305) with existing information on the base content of codons and abundance of amino acid in different organisms. A comparison is also made between the three groups of amino acids suggested by the model and the two classes of aminoacetyl-tRNA synthetases. The observed agreements tend to suppot the model.

Gene Unscrambler for detangling scrambled genes in ciliates

UNLABELLEDnScrambled genes are surprisingly common in some species of ciliates. Until now there was no software available to analyze automatically these genes. We present here a program that can automatically align the macronuclear and micronuclear forms of a gene, outputting the location of the macronuclear destined segments and pointer sequences.nnnAVAILABILITYnA web version of the program is available free of charge and can be accessed at http://oxytricha.princeton.edu/GeneUnscrambler.htm

On the Classes of Aminoacyl-tRNA Synthetases, Amino Acids and the Genetic Code

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering modelsfor the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporationof new amino acids following duplication and divergence of previoussynthetases and tRNA molecules.