Germline nuclei contain more DNA than somatic nuclei. Flow-cytometric analyses of nuclear DNA content reveal major differences in nuclear DNA content between germline (spermatids) and somatic tissues. Sperm and blood nuclei were isolated from the same four individuals. This figure shows sperm and blood traces from two different animals. Blue and red arrows mark the relative sizes of 1C sperm (2.31 pg) and 2C blood (1C = 1.82 pg) genomes, relative to arbitrary fluorescence units. Nuclei from each tissue and individual were measured in separate runs along with trout erythrocyte nuclei (TEN) as internal standards. An asterisk marks a peak corresponding to 2C spermatogonia or sperm doublets. (Left) The small blue peak under the (red) blood peak likely indicates a minor amount of somatic cells in the testes sperm preparation (see also Fig. S1). (Right) Somatic cells are less apparent in the sperm trace.

Germ1. (A) Sequence analysis: Germ1 was isolated by screening a size-selected (9–10 kb) HindIII library (from sperm DNA) for presence of the rpt200 probe. Sequencing of this fragment revealed that this particular germline-enriched element is internally composed of several different repetitive elements. Analysis of somatic depth of coverage reveals that much of the sequence is abundantly represented by somatic whole genome shotgun reads, similar to the functional rDNAs. Notably though, the 5′ region (not homologous to 28S RNA) is relatively rarer. Labels show the position of sequence elements and molecular probes: FISH, the FISH probe corresponding to the somatically rare region (see Fig. S3); RT, the real-time probe for Germ1 (note that this probe overlaps the boundary between somatically-rare and 28S rDNA-like regions); RT (control), the real-time probe for 28S rDNA (note that the forward primer matches only rDNA, and the reverse primer matched both sequences) (see Fig. 4). (B) Structural differences between germline and soma are widespread and reproducible. To test whether fragments that were identified as sperm specific in our initial screen are truly unique to germline, rpt200 was used to probe Southern blots of respective genomic DNAs that were isolated from testes and several somatic tissues of two animals. Fragment sizes were estimated on the basis of electrophoretic migration of molecular mass markers (Low Range PFG Marker; New England Biolabs) that were run in adjacent lanes. T, testes; B, blood; L, liver; K, kidney; F, fin; M, muscle.

The distribution of Germ1-like sequences differs strikingly between germline and somatic genomes. Germ 1 strongly hybridizes to eight germline (testes) chromosomes (meiotic metaphase I), whereas hybridization to somatic (gill) chromosomes (mitotic metaphase) from the same animal is limited to a single chromosome pair. These hybridizations show clear changes in genome structure and Germ1 copy number.

Variation in abundance of Germ1 over embryonic development. The abundance of Germ1 was measured relative to 28S rDNA. Data are plotted relative to the log ratio of these two fragments in sperm DNA. These measurements indicate that loss of the Germ1 fragment occurs between 2 and 3 days postfertilization, at approximately the transition from blastula to gastrula. Real-time probes were designed to specifically detect Germ1 or canonical rDNAs; their locations are shown in Fig. 2. A timeline of major embryological events (16) is superimposed on this plot. Corresponding Tahara (34) stages are (formatted as day:stage): D2:11, D3:14, D4:18, D6:21, D8:22, D10:23, D12:24, D14:25.

A lamprey SPOPL homolog is present and expressed in the germline but lost from soma during embryonic development. (A) Polymerase chain reactions were performed using primers that were targeted to a lamprey SPOPL homolog and an internal control (IC). Templates were genomic DNA (gDNA) from sperm (S) and blood (B), cDNA from adult (A) and juvenile (J) lamprey, RNAs from these same samples, and diH₂O (X). Amplifications of genomic DNAs show that the gene is present in sperm but substantially reduced in blood. Amplifications of cDNAs reveal expression of the gene in juvenile and adult testes. Failure to amplify fragments from source RNAs rules out the possibility that contaminating genomic DNAs contributed to the amplification of SPOPL fragments from cDNAs. Amplified fragments are flanked by size standards (M, 1 kb Plus DNA Ladder; Invitrogen). (B) Variation in abundance of SPOPL over embryonic development. The abundance of SPOPL was measured relative to a single-copy gene HMG. Data are plotted relative to the log ratio of these two fragments in sperm DNA. The value for blood is plotted but is not distinguishable from zero. These measurements indicate that loss of SPOPL occurs during early development and is already apparent by day 2 postfertilization. A timeline of major embryological events (16) is superimposed on this plot. See Fig. 4 for corresponding Tahara (34) stages.