Functional and comparative analysis of globin loci in pufferfish and humans

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2842-2849 ◽  
Author(s):  
Nynke Gillemans ◽  
Tara McMorrow ◽  
Rita Tewari ◽  
Albert W. K. Wai ◽  
Carola Burgtorf ◽  
...  
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Cell Types ◽  
Specific Cell ◽  
Fish Analysis ◽  
Globin Genes ◽  
Site Mapping ◽  
Physical Linkage ◽  
Human And Mouse

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing α- and β-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show thatFugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only α-globin genes (α-locus), whereas the other also contains a β-globin gene (αβ-locus). This is the first poikilothermic species analyzed in which the physical linkage of the α- and β-globin genes has been uncoupled, supporting a model in which the separation of the α- and β-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu αβ-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian β-globin clusters, may not be present in the Fugu αβ-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

scholarly journals Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

2019 ◽  
Author(s):  
Arnav Moudgil ◽  
Michael N. Wilkinson ◽  
Xuhua Chen ◽  
June He ◽  
Alex J. Cammack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Single Cell ◽  
Binding Sites ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Specific Cell

AbstractIn situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

Different distributions of homologous chromosomes in adult human Sertoli cells and in lymphocytes signify nuclear differentiation

1996 ◽  
Vol 109 (4) ◽  
pp. 773-776 ◽  
Author(s):  
A.C. Chandley ◽  
R.M. Speed ◽  
A.R. Leitch
Keyword(s):  
Sertoli Cells ◽  
Cell Types ◽  
Fish Analysis ◽  
Cell Nuclei ◽  
Interphase Nuclei ◽  
Homologous Chromosomes ◽  
Blood Lymphocytes ◽  
Painting Probes ◽  
Whole Chromosome Painting

Using whole chromosome painting probes for human chromosomes 3,7,8,13,17 and 21 and X and the probe pHY2.1 for the Y chromosome coupled with fluorescent in situ hybridization (FISH) analysis, the distribution of chromosomes is reported in nuclei of Sertoli cells of the adult testis and in stimulated blood lymphocytes. The distribution of chromosomes in the two cell types is significantly different. A strong tendency for each pair of homologues to pair is inferred from the observation of only a single detectable signal in the majority of Sertoli cell nuclei. The sex chromosomes, by contrast, give two clearly separated signals. Interphase nuclei in dividing blood lymphocytes, analysed as controls, also show mainly two separated signals for all non-acrocentric autosomal pairs, but acrocentric pairs no. 13 and 21 show some tendency to associate, probably reflecting satellite association.

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 543-554 ◽  
Author(s):  
A.L. Brice ◽  
J.E. Cheetham ◽  
V.N. Bolton ◽  
N.C. Hill ◽  
P.N. Schofield
Keyword(s):  
Growth Factor ◽  
Cell Types ◽  
Specific Cell ◽  
Insulin Like Growth Factor ◽  
Vitro Fertilization ◽  
Age Related ◽  
Factor Ii ◽  
Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1041-1049 ◽  
Author(s):  
A. Leder ◽  
A. Kuo ◽  
M.M. Shen ◽  
P. Leder
Keyword(s):  
In Situ Hybridization ◽  
Embryonic Development ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Globin Mrna ◽  
Mature Adult ◽  
Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

scholarly journals The -175T----C mutation increases promoter strength in erythroid cells: correlation with evolutionary conservation of binding sites for two trans-acting factors

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 756-761 ◽  
Author(s):  
DL Gumucio ◽  
WK Lockwood ◽  
JL Weber ◽  
AM Saulino ◽  
K Delgrosso ◽  
...  
Keyword(s):  
Binding Site ◽  
Point Mutation ◽  
Binding Sites ◽  
Globin Gene ◽  
Evolutionary Conservation ◽  
Single Point Mutation ◽  
Promoter Strength ◽  
Globin Genes ◽  
Gamma Globin ◽  
Specific Distribution

Abstract A point mutation at position -175 has been detected in Agamma as well as Ggamma globin genes in individuals with hereditary persistence of fetal hemoglobin (HPFH). To prove that this single point mutation results in increased promoter strength, we transfected erythroid and nonerythroid cell lines with constructs containing normal and mutant promoters linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Differences in transfection efficiency were controlled by cotransfection of pRSVgpt. In K562 erythroleukemia cells, the -175 HPFH promoter directed three- to fourfold more CAT activity than its wild type counterpart. However, in HeLa cells the two promoters were similar in strength. The -195 to -165 region of the gamma-globin promoter contains binding sites for two proteins: a ubiquitously distributed octamer binding protein, OBP, and the erythroid-specific protein, GF-1. We find that while the GF-1 binding site is highly conserved among related primate gamma-globin genes, the octamer binding site is not. The evolutionary conservation of GF-1 as well as its erythroid-specific distribution suggest that this protein is important in gamma-globin gene expression. A role for OBP in the regulation of gamma-globin, if any, must have arisen recently in primate evolution.

scholarly journals Single-Cell Transcriptomic Map of the Human and Mouse Bladders

2019 ◽  
Vol 30 (11) ◽  
pp. 2159-2176 ◽  
Author(s):  
Zhenyuan Yu ◽  
Jinling Liao ◽  
Yang Chen ◽  
Chunlin Zou ◽  
Haiying Zhang ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Epithelial Cells ◽  
Single Cell ◽  
Cell Types ◽  
Specific Cell ◽  
Human Bladder ◽  
Healthy Human ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Human And Mouse

BackgroundHaving a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer.MethodsWe used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions.ResultsWe created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders.ConclusionsThis transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

scholarly journals Methods to Enhance Signal Using Isotopic In Situ Hybridization

2002 ◽  
Vol 50 (8) ◽  
pp. 1031-1037 ◽  
Author(s):  
Betty Ky ◽  
Paul J. Shughrue
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Oxytocin Receptor ◽  
Specific Cell ◽  
Rat Hypothalamus ◽  
Low Levels ◽  
Tuberoinfundibular Peptide ◽  
Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

scholarly journals Marine Planktonic Archaea Take Up Amino Acids

2000 ◽  
Vol 66 (11) ◽  
pp. 4829-4833 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
Amino Acids ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Similar Proportion ◽  
Specific Cell ◽  
Natural Conditions ◽  
The Pacific ◽  
The Pacific Ocean ◽  
Archaeal Communities

ABSTRACT Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.

scholarly journals Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0 identifies proteins enriched within inflammatory lesions in tissue sections and human clinical biopsies.

2021 ◽  
Author(s):  
BOCHENG YIN ◽  
Laura R Caggiano ◽  
Rung-Chi Li ◽  
Emily McGowan ◽  
Jeffery W. Holmes ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Biology ◽  
Cell Types ◽  
Specific Cell ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Novel Approach ◽  
Esophageal Tissue ◽  
Inflammatory Proteins

Tissue microenvironment properties like blood flow, extracellular matrix or proximity to immune infiltrate are important regulators of cell biology. However, methods to study regional protein expression in context of the native tissue environment are limited. To address this need we have developed a novel approach to visualize, purify and measure proteins in situ using Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0. We previously implemented AutoSTOMP to identify proteins localized to the vacuoles of obligate intracellular microbes at the 1-2 μm scale within infected host cells1. Here we report custom codes in SikuliX to specify regions of heterogeneity in a tissue section and then biotin tag and identify proteins belonging to specific cell types or structures within those regions. To enrich biotinylated targets from fixed tissue samples we developed a biochemical protocol compatible with LC-MS. These tools were applied to a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and b) characterize inflammatory proteins enriched in IgG4+ lesions in esophageal tissue. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools are limited.

scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D. Garcia ◽  
Emma R. West ◽  
Jiho Choi ◽  
Sylvain W. Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

ABSTRACTRecent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

Sign in / Sign up

Export Citation Format

Share Document