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I know definition of the disease pygopagus but I want to know the meaning of separate parts of it, in fact what's the meaning of pygo- and -pagus in terminology?
According to Wiktionary.com:
- pygo- means "rump" or "posterior" from Ancient Greek πυγή (pugḗ, “tail, rump”).
According to dictionary.com:
- -pagus means "fixation" or "something fixed or solid" from Greek págos.
So literally, pygopagus (or conjoined twins attached at the butt) means "fixed rumps".
David A. Staffenberg MD, DSc (Hon) , James T. Goodrich MD, PhD, DSc(Hon) , in Plastic Surgery Secrets Plus (Second Edition) , 2010
6 How did conjoined twins become known as “Siamese twins”?
Conjoined twins throughout history have captivated people. They have been worshipped as gods or feared as bad omens, leading them to be abandoned, exiled, or even killed. As time passed, they were viewed as curiosities, and those who survived became sideshow acts, performed in circuses, or even became stage performers. Until the late 1800s conjoined twins were called “monsters.” The term Siamese twins comes from the twin conjoined brothers Chang and Eng Bunker who were born in Siam, now Thailand. When they first arrived in England to become circus exhibits, they were called “The Siamese Twins.”
Conjoined Twins: Definition and Statistics
* A woman only produces one single egg, which does not fully separate after fertilization * Developing embryo starts to split into identical twins during the first few weeks after conception but the process stops before its complete * Another theory suggests that two separate embryos may somehow fuse together in early development
* There aren’t any specific signs and symptoms that a woman is carrying conjoined twins * As with other twin pregnancies, the uterus may grow more rapidly than expected, and mothers of twins may also have more fatigue, nausea and vomiting early in the pregnancy * Conjoined twins are classified by where they are joined * Medical experts use words to identify conjoined twins that contain “pagus” meaning “fastened” in Greek * Thoracopagus twins are joined at the chest, share a heart and may also share a liver and upper intestine * Omphalopagus twins are joined near the bellybutton, share the liver, and some share the lower part of the small intestine (ileum) and colon * Pygopagus twins are joined at the base of the spine, share the lower gastrointestinal tract, and a few share the genital and urinary organs * Ischiopagus twins are joined at the pelvis, share the lower gastronintestinal tract, as well as the genital and urinary tract organs * Craniopagus twins are joined at the heard, share a skull and possibly brain tissue, some also share cerebral cortex—the part of the brain that plays a central role in memory, language and perception.
Twins are defined as two offspring resulting from the same pregnancy. This is a not uncommon pregnancy outcome in humans and can occur naturally either through the division of a single fertilized egg (monozygotic) or two fertilized eggs (dizygotic). Monozygotic (MZ) twinning is less frequent than dizygotic (DZ) twinning, with an estimated incidence of 3 MZ twins per 1,000 pregnancies (Bortolus et al., 1999 ), a rate that is consistent across different populations. In contrast, the rate of DZ twinning shows significant differences in different populations. In the United States, the rate of twin pregnancies nearly doubled in the 30 years beginning in 1980 (Martin, Hamilton, & Osterman, 2012 ), likely being driven by assisted reproductive technologies, with the current rate estimated to be 16.7 twin sets per 1,000 pregnancies. The highest rate of twin pregnancies is seen in the Yoruba people of Nigeria with 45–50 twin sets per 1,000 pregnancies. In contrast, the lowest rates of twinning are found in South (India, Pakistan, Nepal, Bangladesh) and Southeast Asia with an estimated 6–9 twin sets per 1,000 pregnancies (Bortolus et al., 1999 ).
Conjoined twins (CTws) result from an anomalous MZ twinning event in early embryonic development. Incidence of CTws is estimated at between 1 in 50,000 and 1 in 100,000 births, with observed female predominance (male-to-female ratio
1:2) across populations (Mutchinick et al., 2011 ). The purpose of this study is to explore the representation of CTws in cultural objects, perform a cross-cultural comparison of the objects, and speculate on the significance of the images in the broader anthropological context of the cultures.
1.1 Conjoined twins
The exact etiopathogenesis of CTws remains unclear. Two popular but contradictory theories have emerged over time to explain the mechanism of development of CTws. The first is the theory of “partial fission,” which postulates that CTws result from a late (typically after Day 15 of embryonic development) and incomplete split of the embryonic disk of normally developing MZ twins (Kaufman, 2004 Sadler, 2010 ). The second theory, the theory of “secondary fusion,” suggests that CTws result from the coalescence and fusion of two initially separate monovular embryonic disks (Spencer, 2000a Spencer, 2000b ). Although CTws are thought to have a genetic basis, no causal genetic variants have been identified.
CTws are classified by the anatomical site of attachment, with the suffix “-pagus,” a Greek word for “fixed” or “fastened” (Table 1). The two major groups are symmetrical and asymmetrical CTws. While symmetrical CTws include both ventral and dorsal unions, asymmetrical CTws have an incomplete twin attached to the other twin at any location (Spencer, 1996 ). No examples of asymmetric CTws in cultural artifacts were identified, so are not considered further.
- Parapagus dicephalus—share the entire trunk with separate heads
- Parapagus diprosopus—one trunk and one head with two faces on the same side of the head
- Parapagus dithoracic—joined at the abdomen and pelvis with separate thoraces
1.2 Cultural significance of twins and congenital anomalies
The interpretation of congenitally anomalous births by world cultures throughout history has not been informed by modern scientific knowledge. Many cultures overlay spiritual meaning to these incidents. In the northeastern region of North America's First Nations peoples, people with dwarfism were often deemed to possess unique spiritual insights. Presumably, the spirits would have a unique role for a person whose body was not suited for other types of work. In Mesoamerica, the condition of kyphosis was associated with the gods, depending on the epoch and the culture. In the Preclassic period, images of Huehueteotl (the old, old fire god) shows him arched far forward holding a brazier on his back, (although whether his posture reflects congenital kyphoscoliosis or age-related kyphosis is unclear). In the Postclassic Huasteca region, the gods of maize and agriculture have the same condition (Richter, 2019 ).
Twinning in cultural art is often connected with spiritual interpretations. The Yoruba culture first viewed twinning as a preternatural occurrence. The divine and shared soul of the twins represented a potential negative in spiritual terms, and early on, twins were often sacrificed. In time, this changed, and cultural and artistic practices embraced and encouraged the advent of twinning retaining a supernatural view of the event. The village Diviner, the Babalawo, would come shortly after a birth to dedicate the twins' spirits to positive forces. In Yoruba art, Ire Ibeji twin effigies were produced if one or both of the twins died (Figure 1). This would serve to rebalance the spiritual unbalance caused by the death. Twinning for the Yoruba has long had a powerful spiritual connection (Leroy, Olaleye-Oruene, Koeppen-Schomerus, & Bryan, 2002 ).
Yoruba Ibeji figures, representing dizygotic twins. From Wellcome Images, a website operated by Wellcome Trust . Attribution 4.0 International (CC BY 4.0)
Native American spiritual legends in North and Mesoamerica feature many twin deities or spiritual actors. The stories range from the creation of the world to good and evil, thunder, and so forth, depending on the region and cultural heritage (Redish & Lewis, 1998a ).
Among the hundreds of gods in the Syro-Hittite pantheon (in Mesopotamia), there are numerous examples of twin gods. Included are the twin goddesses meant to represent the twin phases of nature—the Queen of Heaven and the Queen of Hades Mashu and Mashtu, male and female twin children of the moon Mitra and Varuna who measured out the span of human life and Ea and Anu gods of the planets (Mackenzie, 1915 ). When the focus is narrowed to CTws, there is much less evidence available to evaluate the sociocultural response to this rare event. As with twins in general the response has ranged from rejection from society to the assigning of spiritual attributes to the phenomenon. In art, CTws seem to show a significant preference for spiritual interpretation.
Do kinases cascade? How well is cell regulation understood? What are the best ways to model regulatory systems? Attempts to answer such questions can have bearings on the way in which research is conducted. Fortunately there are recurring themes in regulatory processes from many different cellular contexts, which might provide useful guidance. Three principles seem to be almost universal: regulatory interactions are cooperative regulatory decisions are made by large dynamic protein complexes and regulation is intricately networked. A fourth principle, although not universal, is remarkably common: regulatory proteins are actively placed where they are needed. Here, I argue that the true nature of cell signalling and our perceptions of it are in a state of discord. This raises the question: Are our misconceptions detrimental to progress in biomedical science?
What is the meaning of pygo and pagus in the word pygopagus? - Biology
Streptococcus pyogenes and Streptococcal Disease (page 1)
Streptococcus pyogenes(Group A streptococcus) is a Gram-positive, nonmotile, nonsporeforming coccus that occurs in chains or in pairs of cells. Individual cells are round-to-ovoid cocci, 0.6-1.0 micrometer in diameter (Figure 1). Streptococci divide in one plane and thus occur in pairs or (especially in liquid media or clinical material) in chains of varying lengths. The metabolism of S. pyogenes is fermentative the organism is a catalase-negative aerotolerant anaerobe (facultative anaerobe), and requires enriched medium containing blood in order to grow. Group A streptococci typically have a capsule composed of hyaluronic acid and exhibit beta (clear) hemolysis on blood agar.
Figure 1. Streptococcus pyogenes. Left. Gram stain of Streptococcus pyogenes in a clinical specimen. Right. Colonies of Streptococcus pyogenes on blood agar exhibiting beta (clear) hemolysis.
Streptococcus pyogenes is one of the most frequent pathogens of humans. It is estimated that between 5-15% of normal individuals harbor the bacterium, usually in the respiratory tract, without signs of disease. As normal flora, S. pyogenes can infect when defenses are compromised or when the organisms are able to penetrate the constitutive defenses. When the bacteria are introduced or transmitted to vulnerable tissues, a variety of types of suppurative infections can occur.
In the last century, infections by S. pyogenes claimed many lives especially since the organism was the most important cause of puerperal fever (sepsis after childbirth). Scarlet fever was formerly a severe complication of streptococcal infection, but now, because of antibiotic therapy, it is little more than streptococcal pharyngitis accompanied by rash. Similarly, erysipelas (a form of cellulitis accompanied by fever and systemic toxicity) is less common today. However, there has been a recent increase in variety, severity and sequelae of Streptococcus pyogenes infections, and a resurgence of severe invasive infections, prompting descriptions of "flesh eating bacteria" in the news media. A complete explanation for the decline and resurgence is not known. Today, the pathogen is of major concern because of the occasional cases of rapidly progressive disease and because of the small risk of serious sequelae in untreated infections. These diseases remain a major worldwide health concern, and effort is being directed toward clarifying the risk and mechanisms of these sequelae and identifying rheumatogenic and nephritogenic strains of streptococci.
Acute Streptococcus pyogenes infections may present as pharyngitis (strep throat), scarlet fever (rash), impetigo (infection of the superficial layers of the skin) or cellulitis (infection of the deep layers of the skin). Invasive, toxigenic infections can result in necrotizing fasciitis, myositis and streptococcal toxic shock syndrome. Patients may also develop immune-mediated post-streptococcal sequelae, such as acute rheumatic fever and acute glomerulonephritis, following acute infections caused by Streptococcus pyogenes.
Streptococcus pyogenes produces a wide array of virulence factors and a very large number of diseases. Virulence factors of Group A streptococci include: (1) M protein, fibronectin-binding protein (Protein F) and lipoteichoic acid for adherence (2) hyaluronic acid capsule as an immunological disguise and to inhibit phagocytosis M-protein to inhibit phagocytosis (3) invasins such as streptokinase, streptodornase (DNase B), hyaluronidase, and streptolysins (4) exotoxins, such as pyrogenic (erythrogenic) toxin which causes the rash of scarlet fever and systemic toxic shock syndrome.
Classification of Streptococci
Hemolysis on blood agar
The type of hemolytic reaction displayed on blood agar has long been used to classify the streptococci. Beta -hemolysis is associated with complete lysis of red cells surrounding the colony, whereas alpha-hemolysis is a partial or "green" hemolysis associated with reduction of red cell hemoglobin. Nonhemolytic colonies have been termed gamma-hemolytic. Hemolysis is affected by the species and age of red cells, as well as by other properties of the base medium. Group A streptococci are nearly always beta-hemolytic related Group B can manifest alpha, beta or gamma hemolysis. Most strains of S. pneumoniae are alpha-hemolytic but can cause ß-hemolysis during anaerobic incubation. Most of the oral streptococci and enterococci are non hemolytic. The property of hemolysis is not very reliable for the absolute identification of streptococci, but it is widely used in rapid screens for identification of S. pyogenes and S. pneumoniae.
The cell surface structure of Group A streptococci is among the most studied of any bacteria (Figure 2). The cell wall is composed of repeating units of N-acetylglucosamine and N-acetylmuramic acid, the standard peptidoglycan. Historically, the definitive identification of streptococci has rested on the serologic reactivity of "cell wall" polysaccharide antigens as originally described by Rebecca Lancefield. Eighteen group-specific antigens (Lancefield groups) were established. The Group A polysaccharide is a polymer of N-acetylglucosamine and rhamnose. Some group antigens are shared by more than one species. This polysaccharide is also called the C substance or group carbohydrate antigen.
Fat content, fatty acid composition and estimates of energy metabolism of adélie penguins (Pygoscelis adeliae) during the early breeding season fast
1. Adélie penguins (Pygoscelis adeliae) breeding at Cape Crozier, Antarctica, arrive in the colony from the sea in October at the beginning of the austral summer. Both sexes fast while on the breeding colony for 3–6 weeks before returning to the sea to feed.
2. Six individuals collected during the first fast period showed decreased blubber thickness and a linearly corresponding decrease in ether extractable fat with time after arrival in the breeding area. Birds contained about 45 per cent of dry weight as fat upon arrival, and in a typical incubation, males decreased to about 20 per cent after day 27.
3. The decrease in fat represents approximately 56 g of fat used per day by fasting male Adélies for the 27-day period. On the basis of this value, it has been estimated that 490 kcal/bird per day approximates energy demands for these fasting birds during the early part of the breeding season.
4. Fatty acid compositions of total ether extractable lipids, subcutaneous fat and abdominal depot fat did not differ significantly except in a few of the long-chain acids. Depot fat fatty acids of normal breeding birds did not change significantly from arrival on the colony to day 27.
5. Proportions of fatty acids in Adélie penguin depot fat correspond with the proportions of fatty acids in their normal diet of krill (Euphausia superba).
What is the meaning of pygo and pagus in the word pygopagus? - Biology
Other styles suggested the letter forms of roman and italic type. Roman type was used by several printers before Nicolas Jenson Jenson or Janson, Nicolas
, d. c.1480, Venetian printer, b. France. Jenson studied printing with Gutenberg at Mainz for three years.
. Click the link for more information. so improved it as to ensure its triumph as the standard type. Italic type was first used by Aldus Manutius Aldus Manutius
or Aldo Manuzio
, 1450, Venetian printer. He was educated as a humanistic scholar and became tutor to several of the great ducal families. One of them, the Pio family, provided him with money to establish a printery in Venice.
. Click the link for more information. , who also introduced small capitals. Roman type is of two basic sorts, old style and modern. The modern type emphasizes the contrast between light and heavy lines and has conspicuous level serifs the old style type keeps its lines of nearly the same weight and has inconspicuous serifs, some of them sloping. Qualities of old style and modern types are often combined. Into the mid-20th cent. type characters were usually made by pouring metal into previously cut matrices and, less frequently, by processes using plastics and other synthetic materials. Computerization of type design and photomechanical printing techniques have almost entirely replaced metal type. By the early years of the 21st cent. the computer had made the design of new styles of type, once an arduous task, a relatively simple process. Tens of thousands of type fonts are now in existence, and new styles of type are created on a nearly daily basis.
Famous designers of types include, in addition to those named above, Geofroy Tory Tory, Geofroy
, c.1480, Parisian printer, typographer, and author, b. Bourges. After study in Italy, he won distinction as a professor in Paris and became editor to the printer Henri Estienne.
. Click the link for more information. , Claude Garamond Garamond, Claude
, 1480, Parisian designer and maker of printing types. According to tradition he learned his art from Geofroy Tory. Types designed by Garamond were used in the printeries of the Estienne family, Colines, Plantin, and Bodoni, and types used by the
. Click the link for more information. , Robert Granjon Granjon, Robert
, fl. 1545, French designer of type and printer. He began his work in Paris and afterward worked in Lyons, Antwerp, and Rome. The types that he designed and made included roman, italic, Greek, Hebrew, and Syriac.
. Click the link for more information. , Christopher van Dyck Dyck, Christopher van
, 1601–c.1672, German designer and maker of printing type, who worked in Amsterdam. Types that he designed were used by the Elzevir firm. His roman typeface was of the kind known in England and America as "old style" and on the Continent as "Elzevir.
. Click the link for more information. , William Caslon Caslon, William
, 1692, English type designer, b. Worcestershire. He worked first in London as an engraver of gunlocks, then set up his own foundry in 1716. The merits of Caslon's types were rediscovered after a brief eclipse in the popularity of John Baskerville's
. Click the link for more information. , John Baskerville Baskerville, John
, 1706, English designer of type and printer. He and Caslon were the two great type designers of the 18th cent. in England. He began his work as printer and publisher in 1757 and in 1758 became printer to the Univ. of Cambridge.
. Click the link for more information. , Giambattista Bodoni Bodoni, Giambattista
, 1740, Italian printer b. Piedmont. He was the son of a printer and worked for a time at the press of the Vatican. Under the patronage of the duke of Parma, he produced stately quartos and folios with impressive title pages and luxurious margins.
. Click the link for more information. , François Ambroise Didot Didot, François
, 1689, Parisian printer. The son of a printer, Denis Didot, he was the first of the family to win fame in his craft. His son, François Ambroise Didot,
. Click the link for more information. , William Morris Morris, William,
1834, English poet, artist, craftsman, designer, social reformer, and printer. He has long been considered one of the great Victorians and has been called the greatest English designer of the 19th cent.
. Click the link for more information. , Bruce Rogers Rogers, Bruce,
1870, American typographer and book designer, b. Lafayette, Ind. As printing adviser to Cambridge Univ. Press, Harvard Univ. Press, and to commercial houses specializing in limited editions and fine printing, he earned a reputation as his era's leading
. Click the link for more information. , F. W. Goudy Goudy, Frederic William
, 1865, American type designer, b. Bloomington, Ill. Goudy is celebrated as one of the finest and most prolific type designers in history. In 1905, Goudy established his first press, which he moved to New York City the next year.
. Click the link for more information. , and the contemporary American Matthew Carter.
See also typography typography
, the art of printing from movable type. The term typographer is today virtually synonymous with a master printer skilled in the techniques of type and paper stock selection, ornamentation, and composition.
. Click the link for more information. .
See F. W. Goudy, Alphabet and Elements of Lettering (repr. 1922) H. Lehmann-Haupt, One Hundred Books about Bookmaking (1949) J. R. Biggs, An Approach to Type (2d ed. 1962) S. Carter, Twentieth-century Type Designers (1987) A. S. Lawson with D. Agner, Printing Types (rev. and expanded ed. 1990) W. P. Jaspert et al., Encyclopaedia of Type Faces (5th ed. 2001) D. B. Updike, Printing Types (4th ed. 2001) P. Baines and A. Haslam, Type and Typography (2002) M. Bierut, Seventy-nine Essays on Design (2007) J. Tholenaar and A. W. Purvis, Type: A Visual History of Typefaces and Graphic Styles (2009). See also bibliography under typography typography
, the art of printing from movable type. The term typographer is today virtually synonymous with a master printer skilled in the techniques of type and paper stock selection, ornamentation, and composition.
. Click the link for more information. .
a rectangular piece of metal, plastic, or wood with a raised image of a letter or character on one side. The raised or recessed image serves to reproduce letters and characters by printing, in which the face is covered with ink and an impression is made on paper. Metal type is the most common it is cast from printing alloy. The parts of a piece of type (see Figure 1) are the body (a), beard (b), and face (c) the dimensions of the type are defined by the point size (d), width (e), and height to paper (f). The last dimension is constant for all kinds of type.
an element with which a particular taxon is always associated. The type of a species or an intraspecific taxon is usually a single specimen of a plant or animal or, less commonly, several specimens viewed together on one herbarium sheet or in one laboratory preparation. Sometimes a drawing serves as a type. The type of the plant species Companula aldanensis is a specimen collected by the Russian botanist V. S. Korzhevin on Aug. 6, 1928, on the bank of the Aldan River in Siberia the specimen is preserved in Leningrad at the herbarium of the V. L. Komarov Botanical Institute of the Academy of Sciences of the USSR.
The term &ldquotype&rdquo is also used as a designation for a lower taxonomic category that is selected as a standard of reference for a higher category. The type of a genus or of a taxon between a genus or species (for example, a subgenus or section) is a particular species. For example, the species Campanula latifolia is the type of the genus Campanula. The type of family or of a taxon between a family and a genus (for example, a tribe or subfamily) is a particular genus. For example, the type genus of the family Campanulaceae is Campanula, a genus established by C. Linnaeus. Taxa higher than a family do not have types.
Alveolar type II cell (AEC2) dysfunction is a primary cause of pathogenesis in many poorly understood lung diseases that lack effective therapeutics. Patient AEC2s are very difficult to isolate and study. Childhood interstitial lung disease (chILD) is a group of monogenic AEC2 diseases which can be caused by autosomal dominant mutations in the surfactant protein C (SFTPC) gene. Generating AEC2s de novo using induced pluripotent stem cell (iPSC) technology would provide novel opportunities to study diseases of the alveolar epithelium, including SFTPC mutations. Described herein are fluorescent reporter lines that enable the first ever isolation of a pure population of live iPSC-derived AEC2s (iAEC2s) for use in disease modeling and drug screening.
SFTPC Reporter hPSC Lines Allow Identification of Putative iAEC2s.
A fluorescent reporter (GFP) was targeted into the endogenous SFTPC locus of human PSC lines. Site-specific TALE nucleases were used to create a double stranded break near the start codon of SFTPC, facilitating homologous recombination of the fluorescent reporter (FIG. 15A). A directed differentiation approach was used that recapitulates the key developmental milestones of the developing embryo to generate lung progenitors from these iPSCs (FIG. 15B).
Human SFTPC+ Cells Derive from NKX2.1+ Progenitor Cells.
tdTomato was targeted into the SFTPC locus of an iPSC line with an NKX2.1-GFP reporter, resulting in a dual reporter, with putative AEC2s expressing both NKX2.1-GFP and SFTPC-tdTomato, permitting the study of human developmental pathways in-vitro (FIG. 16).
iAEC2s Express Distal Lung mRNA and Protein.
After NKX2.1+ progenitor cells were exposed to distalizing media for 20 days, SFTPCtdTomato+ cells were sorted to purity and analyzed by RT-qPCR, showing expression of AEC2-specific genes at levels similar to or higher than primary week 21 fetal SFTPC+ cells. They also express ABCA3, an important AEC2 lamellar body protein (FIG. 17).
Distal Lung Organoids are Phenotypically Similar to Mature AEC2s.
Since these SFTPCtdTomato+ cells could represent a range of alveolar developmental stages, it was next sought to assess the maturity level of these cells by evaluating whether they express lamellar bodies, a key AEC2 organelle (FIGS. 18A-18B). Expression of 8 kD surfactant protein B (SFTPB) is also a marker of maturity in AEC2s. AEC2s can transdifferentiate into type I alveolar cells (AEC1s) in-vivo in response to injury as well as in-vitro after monolayer culture in DMEM+10% FBS (FIG. 19).
Demonstrated herein is a working reporter iPSC line that facilitates identification and characterization of pure populations of iAEC2s. Human SFTPC+ distal lung cells derive from Day 15 SFTPC− NKX2.1+ cells. Distal iAEC2 organoids express both AEC2 mRNA and protein, as well as lamellar bodies and mature SFTPB protein. They can also upregulate AEC1 markers in response to monolayered culture, suggesting a relatively mature phenotype. iAEC2 organoids represent an in-vitro platform for alveolar disease modeling.
Plant Material and Growth Conditions
Arabidopsis thaliana mutant seed stocks used were in Landsberg erecta ( Ler) and Col genetic backgrounds and were obtained from public stock centers and personal donations. Monogenic mutants were described previously: fve-1, fca-1, ft-1, co-2, and gi-3 ( Koornneef et al., 1991), GA-deficient ga1-3 and ga2-1 ( Koornneef and van der Veen, 1980), soc1-1 ( Samach et al., 2000), ebs ( Piñeiro et al., 2003), tfl2-1 ( Larsson et al., 1998), atx1-2 ( Pien et al., 2008), atxr7-1 and atxr7-2 ( Tamada et al., 2009), elf7-2 ( He et al., 2004), elf6-4 ( Jeong et al., 2009), and axe1-5 ( Yu et al., 2011). clf-16 was previously isolated in our laboratory. The shl-1 allele, in Col background, corresponds to line SALK_053996, and shl-2 allele, in Ler background, corresponds to line GT442, obtained from Cold Spring Harbor Laboratory. Molecular markers used for the genotyping of double mutants are detailed in Supplemental Table 1.
The transcriptional fusions of the SHL and EBS promoters to β-glucuronidase (GUS) (EBSpro:GUS and SHLpro:GUS 2 and 1 kb, respectively) were transformed into Col plants. For the generation of the EBSpro:Myc-EBS construct, the chimeric Myc-EBS cDNA, previously in pGWB18, was cloned into the EBSpro:GUS plasmid by substituting the GUS gene by the Myc-EBS cassette. To generate the SHLpro:Myc-SHL construct in the pGreen0229 plasmid, the Myc-SHL cassette in pGWB18 was cloned downstream of the SHL promoter. The primers used are listed in the Supplemental Table 1. Agrobacterium tumefaciens (AGL0)-mediated transformation of Arabidopsis plants was performed using the floral dip method ( Clough and Bent, 1998). Transformant plants were selected on germination medium (Murashige and Skoog medium with 1% sucrose) ( Murashige and Skoog, 1962) with appropriate antibiotics.
Phenotypic Characterizations and Genetic Analyses
Flowering time, measured as total leaf number, and the duration of vegetative developmental phases were scored as previously described in ASL-Ibercex and Aralab walk-in growth chambers ( Lázaro et al., 2008). Double mutants were isolated from selfed F2 progenies derived from crosses of shl-2 or ebs with different flowering time mutants. To generate double mutants of shl-2 or ebs with flowering time mutants in Col background, shl-2 or ebs was previously introgressed. The molecular markers used for selection are listed in Supplemental Table 1.
Isolation of total RNA from seedlings and cDNA synthesis were performed according to previously described procedures ( Lázaro et al., 2008). Total RNA was extracted from whole seedlings grown under SD for the times indicated. For SHL, CO, FLC, MAF1-5, GA5, FT, and TSF genes, we performed RT-PCR followed by radioactive detection according to described procedures ( Lázaro et al., 2008) (the primers used are described in Supplemental Table 1 ). UBIQUITIN10 (UBQ10) was used as a loading control in these experiments and was amplified for 25 cycles. SHL, FT, TSF, GA5, FLC, and MAF1-5 were amplified for 30 cycles. CO was amplified for 28 cycles. SHL expression was analyzed by RNA gel blot, using as a probe a specific fragment from the 3′ end of the cDNA generated by PCR using the primers SHLnthF (5′-AAACGACGACTTCTTCTGTCG-3′) and SHLnthR (5′-TGAGAAACCA CCATACGCTATAC-3′). SHL expression was also monitored by RT-PCR ( Supplemental Table 1 ). FVE, FLC, and SOC1 expression was analyzed by RNA gel blot. FVE expression was detected using the complete FVE cDNA (1.9 kb) as a probe. FLC and SOC1/AGL20 were detected as described ( Piñeiro et al., 2003). All experiments were repeated at least three times with independent samples.
Transcriptomic analyses were performed on ATH1 arrays using RNA from 18-d-old seedlings grown in SD and harvested at Zeitgeber time 8. Three independent biological replicates were hybridized. We used The Bio-Array Resource for Plant Biology (http://bar.utoronto.ca/welcome.htm), the resources of Genevestigator (https://www.genevestigator.com/gv/), as well as Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html) software for microarray data analysis. Obtained raw data have been submitted to the Gene Expression Omnibus public repository with reference GSE33270.
Histochemical GUS Assays
GUS staining was performed as previously described ( Lázaro et al., 2008).
Three-dimensional models for the EBS-PHD and SHL-PHD domains were generated using the SWISS-PROT/TrEMBL tool (http://swissmodel.expasy.org/) described by Schwede et al. (2003).
Protein Expression and Protein Interaction Assays
The EBS and SHL complete cDNAs together with the fragments corresponding to the PHD domains of both proteins (E-PHD and S-PHD, respectively) were fused to glutathione S-transferase (GST) by cloning them into the pGEX 2T vector compatible with the Gateway system and expressed in Escherichia coli BL21 Rosetta. The primers used are listed in Supplemental Table 1. To generate the EBS-GST and SHL-GST mutated versions, the QuikChange site-directed mutagenesis kit (Stratagene) was used. Nuclear extracts enriched in histones were prepared from MM2d Arabidopsis suspension-cultured cells according to described procedures ( de la Paz Sanchez and Gutierrez, 2009).
For histone pull-down assays, EBS-GST, SHL-GST, E-PHD-GST, S-PHD-GST, and the mutated versions of the proteins bound to glutathione sepharose beads (Upstate) were incubated with histone extracts according to described procedures ( de la Paz Sanchez and Gutierrez, 2009). Pulled down histones were analyzed by immunoblot. For binding assays to the N-terminal tail of H3 methylated at K4, we used biotinylated histone peptides H3K4me0 (12-357), H3K4me1 (12-563), H3K4me2 (12-460), and H3K4me3 (12-564) from Upstate. The proteins E-PHD-GST, S-PHD-GST, and the corresponding mutated versions bound to glutathione sepharose beads were incubated with 0.5 μg of each peptide according to described procedures ( de la Paz Sanchez and Gutierrez, 2009). Bound peptides to different versions of the PHDs were detected with horseradish peroxidase conjugated to Streptavidin after transfer of proteins to Immobilon (Millipore) membranes. For EBS-HDAC pull-down assays, EBS-GST protein was incubated with maltose binding protein fusions to HDA6 and 19 in the same conditions described above for binding assays to histone peptides. Pulled down proteins were visualized with anti-GST antibodies.
For co-IP assays, the different coding sequences were cloned into Gateway destination vectors pGWB18 (C-Myc fusions with EBS and SHL) and pEARLEYGATE201 (HA fusion to HDA6). Agrobacterium strain AGL0 carrying the different constructs was used to infiltrate Nicotiana benthamiana leaves. Co-IP was performed as previously described ( Yu et al., 2011) using anti-HA High Affinity antibody (Roche). Proteins were visualized by immunoblot using an anti-Myc antibody (Millipore).
Coding sequences of the different proteins were cloned into the Gateway binary destination vectors pNXGW (nYFP-) and pXCGW (-cCFP) for BiFC assays. EBS and SHL were tagged with cCFP, and HDA6 was tagged with nYFP at either the N or C terminus, respectively. Assays were performed as previously described ( Yuan et al., 2013). The BiFC constructs were introduced in N. benthamiana leaves by agroinfiltration. YFP-derived fluorescence was analyzed by laser scanning microscopy using a Leica TCS SP8 confocal microscope.
After chromatin isolation according to previously described methods ( Lázaro et al., 2008), the H3 acetylated fractions were immunoprecipitated using specific antibodies to acetylated K9 and K14 (ref. 06-599 from Upstate Biotechnology). PCR was used to amplify four different fragments of the FT gene and three different fragments of the SOC1 gene ( Bouveret et al., 2006) (the primers used are described in Supplemental Table 1 ). All PCR reactions and quantification of the amplified DNA were done as described ( Lázaro et al., 2008). We conducted three repeats of each experiment from independent biological replicates. ACTIN2 was used as an internal control for the ChIP analyses.
Binding of SHL to the SOC1 gene was analyzed by ChIP experiments with pSHL:Myc-SHL plants quantitative PCR was performed on the immunoprecipitates obtained with anti-Myc antibodies with the three primer sets used for ChIP experiments performed with anti-H3 modifications ( Supplemental Table 1 ). Error bars correspond to sd of the mean of at least three quantitative PCR replicates. To establish the binding of EBS to the FT gene, quantitative PCR was performed on the immunoprecipitates obtained with anti-Myc antibodies from pEBS:Myc-EBS plants by using the four primer sets used for ChIP experiments performed with anti-H3 modifications ( Supplemental Table 1 ). Error bars correspond to sd of the mean of at least three quantitative PCR replicates.
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL under the following accession numbers: Al-EBL1 (XP_002868884.1), Al-EBL2 (XP_002872686.1), Th-EBL (BAJ33950.1), Br-EBL1 (EM:DK463881), Br-EBL2 (EM:DY020946), Os-EBL (BAC79935.1), Pt-EBL (XP_002305450.1), Vv-EBL (CBI38025.3), Gm-EBL (ACU15947.1), Zm-EBL (NP_001151899.1), Sb-EBL (XP_002459456.1), Pp-EBL (XP_001781596.1), CO (AT5G15840), EBS (At4g22140), FLC (At5g10140), FT (AT1G65480), FVE (AT2G19520), GA5 (AT4G25420), MAF1 (AT1G77080), MAF2 (AT5G65050), MAF3 (AT5G65060), MAF4 (AT5G65070), MAF5 (AT5G65080), SHL (At4g39100), SOC1 (AT2G45660), TSF (AT4G20370), UBQ10 (AT4G05320), At-ING1 (At3g24010), At-ING2 (At1g54390), Hs-ING2 (AAQ13674.1), Hs-BPTF (NP_872579.2), and GSE33270 (arrays of shl and ebs mutants).
The following materials are available in the online version of this article.
Supplemental Figure 1. SHL Is Expressed at Constant Levels along the Daily Cycle and at Different Times of Development.
Supplemental Figure 2. Flowering Time Phenotype of the Double Mutants shl ebs Grown under LD.
Supplemental Figure 3. The Expression of the Floral Integrator FT and Representative Genes from the Different Pathways Controlling Flowering Time Is Independent of SHL.
Supplemental Figure 4. EBS Binds Genomic Regions of FT but Not of SOC1, While SHL Binds the SOC1 Locus but Not FT.
Supplemental Figure 5. Flowering Time Phenotype of the Double Mutants Combining ebs and shl-2 with Mutations in Genes Encoding Chromatin Remodeling Factors Related with the Levels of H3K4me3.
Supplemental Figure 6. Flowering Time Quantification of the Double Mutants Combining ebs and shl with axe1-5.
Supplemental Figure 7. SHL Interacts in Vivo with HDA6.
Supplemental Table 1. Primers Used in This Work.
Supplemental Data Set 1 . Transcriptomic Profiling of shl and ebs Mutants.