The marrow cell continuum: stochastic determinism.

Traditional models of hematopoiesis have been hierarchical in nature. Over the past 10 years, we have developed data indicating that hematopoiesis is regulated in a continuum with deterministic and stochastic components. We have shown that the most primitive stem cells, as represented by lineage negative rhodamine(low) Hoechst(low) murine marrow cells are continuously or intermittently cycling as determined by in vivo BrdU labeling. When marrow stem cells are induced to transit cell cycle by in vitro exposure to cytokines, either IL-3, IL-6, IL-11, and steel factor or thrombopoietin, FLT3 ligand, and steel factor, they progress through cycle in a highly synchronized fashion. We have determined that when the stem cells progress through a cytokine stimulated cell cycle the homing, engraftment, adhesion protein, global gene expression, and hematopoietic differentiation phenotypes all change in a reversible fashion. This has led to the continuum model, in which, with cycle transit, chromatin is continually changing altering open transcription areas and providing a continually changing landscape of transcriptional opportunity. More recently, we have extended the changing differentiation profiles to differentiation into lung cells and found that non-hematopoietic differentiation also shows cycle related reversibly modulation. These observations all together support a continuum model of stem cell regulation in which the phenotype of the marrow stem cells is continually and reversibly changing over time

Homing and Long-Term Engraftment of Long- and Short-Term Renewal Hematopoietic Stem Cells

Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change

Maize haplotype with a helitron-amplified cytidine deaminase gene copy

BACKGROUND: Genetic maps are based on recombination of orthologous gene sequences between different strains of the same species. Therefore, it was unexpected to find extensive non-collinearity of genes between different inbred strains of maize. Interestingly, disruption of gene collinearity can be caused among others by a rolling circle-type copy and paste mechanism facilitated by Helitrons. However, understanding the role of this type of gene amplification has been hampered by the lack of finding intact gene sequences within Helitrons. RESULTS: By aligning two haplotypes of the z1C1 locus of maize we found a Helitron that contains two genes, one encoding a putative cytidine deaminase and one a hypothetical protein with part of a 40S ribosomal protein. The cytidine deaminase gene, called ZmCDA3, has been copied from the ZmCDA1 gene on maize chromosome 7 about 4.5 million years ago (mya) after maize was formed by whole-genome duplication from two progenitors. Inbred lines contain gene copies of both progenitors, the ZmCDA1 and ZmCDA2 genes. Both genes diverged when the progenitors of maize split and are derived from the same progenitor as the rice OsCDA1 gene. The ZmCDA1 and ZmCDA2 genes are both transcribed in leaf and seed tissue, but transcripts of the paralogous ZmCDA3 gene have not been found yet. Based on their protein structure the maize CDA genes encode a nucleoside deaminase that is found in bacterial systems and is distinct from the mammalian RNA and/or DNA modifying enzymes. CONCLUSION: The conservation of a paralogous gene sequence encoding a cytidine deaminase gene over 4.5 million years suggests that Helitrons could add functional gene sequences to new chromosomal positions and thereby create new haplotypes. However, the function of such paralogous gene copies cannot be essential because they are not present in all maize strains. However, it is interesting to note that maize hybrids can outperform their inbred parents. Therefore, certain haplotypes may function only in combination with other haplotypes or under specialized environmental conditions

A High Resolution Genetic Map Anchoring Scaffolds of the Sequenced Watermelon Genome

As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F8 population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ∼800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits

The Cotton Centromere Contains a Ty3-gypsy-like LTR Retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species

Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB–type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle

Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination

An 11-bp Insertion in Zea mays fatb Reduces the Palmitic Acid Content of Fatty Acids in Maize Grain

The ratio of saturated to unsaturated fatty acids in maize kernels strongly impacts human and livestock health, but is a complex trait that is difficult to select based on phenotype. Map-based cloning of quantitative trait loci (QTL) is a powerful but time-consuming method for the dissection of complex traits. Here, we combine linkage and association analyses to fine map QTL-Pal9, a QTL influencing levels of palmitic acid, an important class of saturated fatty acid. QTL-Pal9 was mapped to a 90-kb region, in which we identified a candidate gene, Zea mays fatb (Zmfatb), which encodes acyl-ACP thioesterase. An 11-bp insertion in the last exon of Zmfatb decreases palmitic acid content and concentration, leading to an optimization of the ratio of saturated to unsaturated fatty acids while having no effect on total oil content. We used three-dimensional structure analysis to explain the functional mechanism of the ZmFATB protein and confirmed the proposed model in vitro and in vivo. We measured the genetic effect of the functional site in 15 different genetic backgrounds and found a maximum change of 4.57 mg/g palmitic acid content, which accounts for ∼20–60% of the variation in the ratio of saturated to unsaturated fatty acids. A PCR-based marker for QTL-Pal9 was developed for marker-assisted selection of nutritionally healthier maize lines. The method presented here provides a new, efficient way to clone QTL, and the cloned palmitic acid QTL sheds lights on the genetic mechanism of oil biosynthesis and targeted maize molecular breeding