Total RNA from 20 defolliculated, enucleated oocytes of a mature female Xenopus tropicalis frog Data were extracted from the CEL files, RMA normalized across all samples and converted to Log2 notation with Partek Genomics Suite (Partek, Missouri, USA). These values were labeled as to their source and the groups compared.

Total RNA from 716 hand-isolated germinal vesicles from oocytes of a mature Xenopus tropicalis frog Data were extracted from the CEL files, RMA normalized across all samples and converted to Log2 notation with Partek Genomics Suite (Partek, Missouri, USA). These values were labeled as to their source and the groups compared.

Total RNA from 1,008 hand-isolated, demembranated germinal vesicles from oocytes of a mature Xenopus tropicalis frog Data were extracted from the CEL files, RMA normalized across all samples and converted to Log2 notation with Partek Genomics Suite (Partek, Missouri, USA). These values were labeled as to their source and the groups compared.

Fluorescent in situ hybridization (FISH) is a technique for determining the cytological localization of RNA or DNA molecules. There are many approaches available for generating in situ hybridization probes and conducting the subsequent hybridization steps. Here, we describe a simple and reliable FISH method to label small RNAs (200-500 nucleotides in length) that are enriched in nuclear bodies in Drosophila melanogaster ovaries, such as Cajal bodies (CBs) and histone locus bodies (HLBs). This technique can also be applied to other Drosophila tissues, and to abundant mRNAs such as histone transcripts.

This paper reports genome sizes of one Hawaiian Scaptomyza and 16 endemic Hawaiian Drosophila species that include five members of the antopocerus species group, one member of the modified mouthpart group, and ten members of the picture wing clade. Genome size expansions have occurred independently multiple times among Hawaiian Drosophila lineages, and have resulted in an over 2.3-fold range of genome sizes among species, with the largest observed in Drosophila cyrtoloma (1C = 0.41 pg). We find evidence that these repeated genome size expansions were likely driven by the addition of significant amounts of heterochromatin and satellite DNA. For example, our data reveal that the addition of seven heterochromatic chromosome arms to the ancestral haploid karyotype, and a remarkable proportion of similar to 70 % satellite DNA, account for the greatly expanded size of the D. cyrtoloma genome. Moreover, the genomes of 13/17 Hawaiian picture wing species are composed of substantial proportions (22-70 %) of detectable satellites (all but one of which are AT-rich). Our results suggest that in this tightly knit group of recently evolved species, genomes have expanded, in large part, via evolutionary amplifications of satellite DNA sequences in centric and pericentric domains (especially of the X and dot chromosomes), which have resulted in longer acrocentric chromosomes or metacentrics with an added heterochromatic chromosome arm. We discuss possible evolutionary mechanisms that may have shaped these patterns, including rapid fixation of novel expanded genomes during founder-effect speciation.

In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with H-3 and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later. (C) 2015 Elsevier Inc. All rights reserved.

We previously demonstrated that the oocyte nucleus (germinal vesicle or GV) of Xenopus tropicalis contains a population of stable RNA molecules derived from the introns of most expressed genes. Here we show that similar stable intronic sequence (sis) RNAs occur in the oocyte cytoplasm. About 9000 cytoplasmic sisRNAs have been identified, all of which are resistant to the exonuclease RNase R. About half have been confirmed as lariat molecules and the rest are presumed to be lariats, whereas nuclear sisRNAs are a mixture of lariat and linear molecules. Cytoplasmic sisRNAs are more abundant on a molar basis than nuclear sisRNAs and are derived from short introns, mostly under 1 kb in length. Both nuclear and cytoplasmic sisRNAs are transmitted intact to the egg at GV breakdown and persist until at least the blastula stage of embryogenesis, when zygotic transcription begins. We compared cytoplasmic sisRNAs derived from orthologous genes of X. tropicalis and X. laevis, and found that the specific introns from which sisRNAs are derived are not conserved. The existence of sisRNAs in the cytoplasm of the oocyte, their transmission to the fertilized egg, and their persistence during early embryogenesis suggest that they might play a regulatory role in mRNA translation.

To compare nuclear and cytoplasmic RNA from a single cell type, free of cross-contamination, we studied the oocyte of the frog Xenopus tropicalis, a giant cell with an equally giant nucleus. We isolated RNA from manually dissected nuclei and cytoplasm of mature oocytes and subjected it to deep sequencing. Cytoplasmic mRNA consisted primarily of spliced exons derived from similar to 6700 annotated genes. Nearly all of these genes were represented in the nucleus by intronic sequences. However, unspliced nascent transcripts were not detected. Inhibition of transcription or splicing for 1-2 d had little or no effect on the abundance of nuclear intronic sequences, demonstrating that they are unusually stable. RT-PCR analysis showed that these stable intronic sequences are transcribed from the coding strand and that a given intron can be processed into more than one molecule. Stable intronic sequence RNA (sisRNA) from the oocyte nucleus constitutes a new class of noncoding RNA. sisRNA is detectable by RT-PCR in samples of total RNA from embryos up to the mid-blastula stage, when zygotic transcription begins. Storage of sisRNA in the oocyte nucleus and its transmission to the developing embryo suggest that it may play important regulatory roles during oogenesis and/or early embryogenesis.