2013 Group 2 Project
- 1 Cytokinesis
- 1.1 Introduction
- 1.2 History
- 1.3 Mechanisms
- 1.4 Microfilament organisation
- 1.5 Animal Cells Vs Plant Cells
- 1.6 Cytokinesis Failure
- 1.7 Current/Future Research
- 1.8 External Links
- 1.9 Glossary
- 1.10 Images
- 1.11 References
Cytokinesis is the process that leads to the production of two daughter cells from one parent. It is achieved by a circumferential ring of differentiated cortex, enriched in actin and myosin, that contracts to divide the cytoplasm. In animal cells this is achieved by constriction of the plasma membrane, whereas in higher plants it is accomplished through a centrifugal process which involves an expanding cell plate that must find and fuse with a predetermined zone of the plasma membrane to produce a new cell wall. In 1902, the first research into the connection between abnormal mitosis (cytokinesis failure) and malignant tumors was introduced by Theodor Boveri and since then much research has been invested to understand the process of cytokinesis, the factors that influence its completion and failure. This page addresses many aspects of the cytokinesis process including its mechanism and functions, a contrast between animal and plant cell cytokinesis as well as the direction of current and future research.
|1882||Cell division had been studied in the 1880s, it was through Walther Flemming that he termed it mitosis due to its mitotic chromosomes.|
|1902||Theodor Boveri was the first to introduce the idea that there might be a connection between abnormal mitosis and malignant tumours. This would later be connected to cytokinesis failure.|
|1961||An American cell biologist named Ray Rappaport pioneered research using physical manipulations of cells to understand the mechanisms of cytokinesis.|
|1971||The contractile ring that surrounds the cleavage furrow is discovered to be made from actin and myosin-2.|
|1971||Microtubule cytoskeleton plays an important role in the choice and positioning of the division site. The position at which the contractile ring assembles is dictated by the mitotic spindle.|
|1972||In a landmark study published in 1972, Thomas Schroeder provided the first ultrastructural description of the contractile ring during cell cleavage in sea urchin eggs. He observed that the contractile ring was composed of microfilaments that were similar to muscle actin filaments. Formation of the contractile ring coincided with the onset of cytokinesis, its spatial location coincided with that of the cleavage furrow and the filaments disappeared when cytokinesis was over, which indicated that the contractile ring might be responsible for cytokinesis.|
|1985||Classic micro-manipulation experiments in echinoderm eggs demonstrated the dynamic nature of this communication by showing that moving the spindle during anaphase causes furrow regression and formation of a new furrow above the spindle midplane.|
|1988||Plant cell cytokinesis usus an expanding cell plate that must find and fuse with a predetermined zone of the plasma membrane to produce a new cell wall.|
|1997||A requirement for Rho, Rac1 and Cdc42. The Rho family of small GTPases regulates changes during cell division during the formation of actin structures beneath the plasma membrane.|
|1998||Discovered zen-4 is required for cytokinesis due to cytokinetic failure of the nematode Caenorhabditis elegans when zen-4 is removed.|
|1999||A simple eukaryotic model for the study of cytokinesis involves fission yeast Schizosaccharomyces pombe.|
|2005||Discovery that the spindle midzone is not needed for a successful cytokinesis. However, the spindle midzone can negatively affect aster-positioned cytokinesis. This negative effect may result from a limited supply of contractile elements.|
|2006||In fission yeast, ∼250 different proteins localize to the division site of cytokinesis.|
|2012||Cytokinesis nodes are organised according to a specific heirachy as a result of the cytokinesis node proteins Rng2 anCdc4, Myo2 and Rlc1 that are responsible for this localisation.|
Assembly of the Central Spindle
In animal cells, the assembly of the central spindle is important in specifying the division plane as well as serving as an early guide for the formation of the midbody in the later stages of cytokinesis. The central spindle is partly derived from the mitotic spindle during early anaphase, as the deactivation of cyclin dependent kinase 1 (CDK1) activity stabilises microtubules and reorganises the mitotic spindle.
Assembly of the central spindle requires some important microtubule associated proteins (MAPs) and complexes including the protein regulator of cytokinesis 1 (PRC1), the centralspindlin complex and the chromosome passenger complex (CPC), among many others.
Protein Regulator of Cytokinesis 1
One of the major microtubule associated proteins involved in bundling and reorganising the microtubules is protein regulator of cytokinesis 1 (PRC1). PRC1 selectively binds to the interface between antiparallel microtubules, inducing bundling and allowing precise localisation of the protein to the centre of the central spindle.
Comprised of mitotic kinesin-like protein 1 (MKLP1) and Rho family GTPase activating protein (GAP) cytokinesis defect family member 4 (CYK-4), the centralspindlin complex is also involved in the bundling of antiparallel microtubules. Like PRC1 it is concentrated to the centre of the central spindle apparatus, where it regulates RhoA and the process of abscission later on during cytokinesis. At the beginning of anaphase, centralspindlin is localised to the plus ends of antiparallel microtubules, initiating bundling and in turn, formation of the central spindle.
Chromosome Passenger Complex
Comprised of Aurora B kinase, INCENP, Survivin, and Borealin, the chromosome passenger complex (CPC) is localised to centromeres during metaphase where it is involved in the attachment of chromosomes to the mitotic spindle. During anaphase however, CPC is relocated to the central spindle where it is involved in the phosphoregulation of redundant central spindle proteins including PRC1 and MKLP1.
Cleavage Plane Specification
The specification of the cleavage plane and its precise positioning between the two groups of separated chromosomes is important so as to prevent chromosome loss. Cleavage plane specification is directed by redundant signals sent by microtubule asters, followed by signals sent from the spindle midzone, to the site of cleavage, otherwise known as the furrow. This signal pattern varies between different cell types and organisms.
Central Spindle Microtubules
The central spindle now plays a critical role in the process of cleavage plane specification, with its microtubules promoting the activation and concentration of the ras homolog gene family member A (RhoA) protein at the equatorial cortex. Central spindle microtubules, along with astral microtubules, emerging from the centrosomes, are believed to help position the point of contraction at the equatorial cortex. This is believed to be carried out through stable microtubule signals – which promote contractility at the equatorial cortex, whilst dynamic microtubules inhibit such signals at the polar region. These dynamic microtubules specifically inhibit Rho activation at all regions in the cortex apart from the equatorial region, thereby limiting contractile activity in the polar regions. There are varying models used to describe the contractile cues received at the cortex as a result of different findings through different cell types and species.
Ras homolog gene family, member A
This is a small Rho family guanosine triphosphatase (GTPase) protein involved in the regulation of cytokinesis. RhoA is responsible in the specification of the cleavage plane and also later on during contractile ring assembly. As mentioned earlier, active RhoA is distributed to the equatorial cortex via positive signals sent from central spindle microtubules. This is done so in a very specific way, deliberately localising RhoA to a narrow zone within the furrow – the site where, ultimately, cleavage will take place. Its activation is dependent on epithelial cell transforming sequence 2 oncogene (Ect2), which also helps in localising it to the furrow. Along with Ect2, RhoA is also dependent on the centralspindlin complex, as previous studies have shown a relationship between spindle positioning and positioning of the cleavage furrow. As centralspindlin accumulates midway between the separated chromosomes, at the centre of the spindle midzone, it provides a perfect reference point upon which RhoA may activate and furthermore localise at the equatorial cortex, forming the cytokinetic furrow.
Contractile Ring Assembly
As with the specification of the cleavage plane, RhoA is heavily involved in the assembly of the contractile ring – an integral, transient component comprised of both actin filaments and myosin II, which, when formed, will contract, causing the plasma membrane to ingress, and ultimately, separate the cytoplasm into two daughter cells. In addition to actin and myosin II working together as filamentous and motor proteins respectively to form the contractile ring, anillin is another protein at work during this process. Anillin binds to both these proteins and others, creating a link between the signals sent from the central spindle and the equatorial cortex.
By activating diaphanous-related formins - proteins which nucleate the formation of unbranched actin filaments, specifically the second isoform of mammalian homolog of Drosophila diaphanous protein (mDia2) – Rho-GTP plays an active role in actin polymerisation and in the stimulation of actin filament assembly. These formins are autoinhibited, but are uninhibited by Rho-GTP binding to it. Once activated, actin binds independently to the cell cortex and concentrates both at the equatorial plane and at the exact site of the formation of the contractile ring by manner of cortical flow.
Along with actin, myosin II, the primary motor protein required for cytokinesis, is recruited to the equatorial plane through RhoA-mediated activation of the rho-kinase (ROCK), a downstream regulator of the Rho family. ROCK activates myosin II firstly by phosphorylation of the myosin regulatory light chain (rMLC) and secondly by phosphorylation of the myosin phosphatase targeting protein (MYPT). Myosin II plays an essential role in contractile ring contraction, to be further explained in Furrow Ingression.
In addition to actin and myosin II, anillin is another protein present at the formation of the contractile ring. This scaffolding protein binds to the filamentous actin protein, myosin II, RhoA and CYK-4, creating a link between the central spindle and the equatorial cortex. In addition to this, anillin also recruits a filament forming GTP-ase to the contractile ring in septin. Septin acts to restrict the zones in which the cleavage furrow is active in, thereby stabilising the contractile ring. Although anillin is not required for furrow formation, it functions to maintain levels of myosin II in the contractile ring and also creates a link between the ring and the plasma membrane.
The contractile ring, having formed at the equatorial cortex, underneath the plasma membrane, must now contract in order for the plasma membrane, with which it is connected, to ingress, dividing the cytoplasm into the two emerging sister cells. The exact constriction mechanism by which this happens is yet to be defined, however, one model in particular has grown in popularity as a result of high intensity imaging techniques such as electron microscopy and total internal reflection fluorescence microscopy revealing the composition of the contractile ring.
The ‘purse-string’ model involves bipolar filaments of myosin II sliding past two antiparallel actin filaments using motor activity, a model similar to the manner in which muscle sarcomeres contract. This model has come under scrutiny as a result of debate over the attachment of the barbed ends of the actin filaments to the plasma membrane through formins.
What has been observed as the furrow ingresses is that as the contractile ring constricts, the cross-sectional area of the ring remains the same, indicating a constant turnover of myosin II and actin.
Formation of the Midbody
As the contractile ring reaches a diameter of about 1-2μm, the central spindle plays a role in keeping the chromosomes apart prior to abscission, the final step in cytokinesis. This is due to the depolymerisation which microtubules undergo in late anaphase.
The midbody is derived directly from the central spindle in almost all systems, with the disassembly of the contractile ring thought to trigger its formation due to the loss of filamentous actin. More than 100 different proteins localise at the midbody, with their function still unclear today, but generally, it is thought that together, they act to provide a target for the abscission mechanisms of cytokinesis, the final stage where the two daughter cells are separated.
The final stage in cytokinesis, abscission is just as complex as the earlier stages as it involves even more proteins for trafficking and membrane fusion. It involves the separation of the previously partitioned daughter cells, which are now joined together by the midbody or intercellular bridge. Golgi derived vesicles, as well as endosome-derived vesicles accumulate next the the midbody, with vesicles already in the midbody fusing with the plasma membrane before abscission.
Endosomal Sorting Complex Required for Transport III (ESCRT-III)
This protein has been found to be an essential abscission factor as it contributes to membrane deformation and cytosolic severing in a variety of biological processes, namely autophagy and virus budding. ESCRT-III localises adjacent to the midbody in late telophase. Spastin – a microtubule severing protein disassembles microtubule bundles inside the midbody by binding to an ESCRT-III associated protein Charged Multivesicular Body Protein 1B (CHMP1B).
Abscission is still poorly understood. It only occurs after the removal of chromatin from the site of division, as the abscission mechanisms could damage chromosomes left unsegregated. Aurora B kinase ensures the coordination between chromosome segregation and cytokinesis.
Microfilaments are responsible for forming a contractile ring around the dividing cells plasma membrane during cytokinesis. This microfilament based contractile ring must be formed at en equal distance from the two corresponding daughter spindles. This is to ensure that after contraction of the ring, a cleavage furrow is developed at the equator allowing simultaneous pinching off of corresponding spindle apparatuses. This ultimately allows the two daughter cells to divide into two equal halves.
The process of microfilament assembly during the cell cycle and leading up to cytokinesis is detailed below:
- As the cell enters prophase, cytoskeleton constituent microfilament fibres are disassembled naturally accompanying the cell growth and rounding that is beginning to occur.
- During cytokinesis microfilaments travel to the equator to form the contractile ring used for cytoplasmic division of the two daughter cells. This region becomes rich in microfilaments and subsequently in myosin II. This maintains the meiotic/mitotic spindle and chromosomes in a peripheral position.
- After cytokinesis the microfilaments then reorganise themselves into stress fibres between related intracellular organelles. This enables the cell to spread on substrates and build up energy sources after the depletion that took place during cell division and subsequent cytokinesis.
Animal Cells Vs Plant Cells
The function of cytokinesis in animal and plant cells is identical. However plant cells possess both a cell wall and a cell membrane where as animal cells only possesses a cell membrane. To compensate for this, the different cells have adopted different processes in order to complete cytokinesis. Cytokinesis begins at different stages of the cell cycle. The cytoplasm of the two separating daughter cells begins to replicate as the respective cells divide. This promptly ensures that both daughter cells receive exactly (or as near as possible) the same amount of genetic material from the mother cell.
|Animal Cell Cytokinesis||Plant Cell Cytokinesis|
Central Spindle Formation
Adenomatous Polyposis Coli
Adenomatous Polyposis Coli (APC), a tumor suppressor gene, is involved in the formation of the mitotic spindle and the proper functioning of the spindle checkpoint. However, cells containing certain mutations of the APC gene, namely the dominant-negative APC gene containing amino acids 1-1,450, that underwent cytokinesis, generally failed.
In cells expressing APC1–1,450, the contact between the cell cortex and microtubule extensions is severely affected, resulting in unanchored spindles and cells with unanchored spindles failed to complete cytokinesis. APC is therefore crucial to the cytokinetic process.
Cytokinesis defect family member 4
An important GTPase Activating Protein (GAP) Cytokinesis defect family member-4 (cyk-4) along with ZEN-4/CeMKLP1, work together in forming the central spindle as well as in regulating the RhoA GTPase protein during division plane specification.
Cyk-4 mutants (constructed in isolation) do not complete cytokinesis. This is due to the incorrect formation of the central spindle, of which cyk-4 is responsible for. One of the primary reasons for this was the formation of the cleavage furrow, where it would form correctly, ingress, but soon after regress.
Cyk-4 is essential to cytokinesis by localising actin and tubilin in both mutant and wild type cyk-4 embryos. Spindles formed during metaphase in the cyk-4 mutant embryos were normal, but during anaphase, significant differences were observed in comparison to the wild type cyk-4 embryos. Microtubule bundles were reduced in size and appeared highly disorganised. Cyk-4 therefore, is required during anaphase for proper formation of the central spindle. It is possible that for this reason, cyk-4 mutant embryos fail to undergo cytokinesis as a result of the failure of the central spindle to be formed.
Through its interaction with both anillin and RhoA – a GTPase protein which regulates actin polymerization, citron kinase (CIT-K) is involved in stabilising the midbody and controlling abscission during cytokinesis.
CIT-K is integral in the process of cytokinesis, but by removing CIT-K and analysing the localisation of central spindle proteins (RACGAP1, ECT2, Aurora B) and of cleavage furrow proteins (actin, myosin IIB, anillin, and RhoA), there was a disparity in the localization of anillin and RhoA in comparison to the control cells. There was also a complete loss of anillin during the late midbody stage at the cytoplasmic bridge in more than two-thirds of the mutated cells.
The same study also found that by deactivating RhoA in late cytokinesis by means of treating the HeLa cells with a toxin (Clostridium botulinum C3-toxin), localisation of anillin was greatly affected, but CIT-K remained the same, meaning that RhoA plays a part in the localisation of anillin at this late point in cytokinesis, but not for CIT-K.
Contractile-ring assembly in fission yeast cytokinesis: Recent advances and new perspectives Contractile-ring assembly in fission yeast cytokinesis: Recent advances and new perspectives
The process of contractile ring assembly durign Cytokinesis was studied on the fission yeast Schizosaccharomyces pombe. Through previous research and the reaserch conducted during this study that cytokinesis nodes are organised according to a specific heirachy as a result of the cytokinesis node proteins Rng2 anCdc4, Myo2 and Rlc1 that are responsible for this localisation. After organisation myosin-II activity and actin dynamics coordinate condensing of thes nodes into a compact contractile ring aswell as subsequent maturation and remodelling before the final constriction that partitions the cell. No significant new information was found in regards to the overall process and proteins that contribute to the assembly and subsequent maturation. However this type fo research allows us to gain a greater underestanding of these protein interaction primarly because at least 130 of these yeasts proteins are found in humans. This understanding will allow scientists to possibly manipulate these proteins in the future if they ave any relation to specific diseases or metabolic processes of interest.
Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis
This study investigated the relationship between the centralsplindin, mitotic spindle and plasma membrane during and subsequent to absicission and how this effects the respective partitioning of segregating daughter chromosomes. It was observed that after midbody absicission the mitotic spindle remains connected to the plasma membrane and remains connected throughout the duration of cytokinesis. The underlying reason for this is not understood but the process was observed and confirmed under view of an electron microscope. It was found that the C1 sectioin of the centralspindlin (Cs) subunit MgcRacGAP interacts with the polyanionic phosphoinositide lipids of the plasma membrane. This centralspindlin is attachet to the mitotic spindle (Ms). This connection facilitates formation of the midzone and midbody through Cs to Ms contraction and provides attachment of the plasma membrane to the midbody during cytokinesis. Only after membrane tethering of centralspindlin does cell division complete.
Polarity sets the stage for cytokinesis Polarity sets the stage for cytokinesis
This study investigated polarity organisation in cells and the effect this has on subsequent cytokinesis completion. Polarity was studied in relation to cell abscission and the segregation of intracellular constituents. It was found that the polarity of different intracellular components (vesicles) was extremly iportant in the partitioning of the cell into outer and inner compartments. Polarity complex proteins were identified as the key factors that determine an intracellular components polarity, in particular cell epithelial tissues. Before absiccission intracellular components were localised to their respective cytokinetic sections of the dividing daughter cells based on their polar propertie. Therefore this plays a major role in enduring each daughter cell divides with an equal amount of intracellular material that can further be manibulated in cytokinesis. It was concluded that polarity does have an influence on spatiotemporal segregation of membrane and vesicle trafficking during later telophase to ensure cytokinesis proceeds correctly.
Does the recommended lymphocyte cytokinesis-block micronucleus assay for human biomonitoring actually detect DNA damage induced by occupational and environmental exposure to genotoxic chemicals? Does the recommended lymphocyte cytokinesis-block micronucleus assay for human biomonitoring actually detect DNA damage induced by occupational and environmental exposure to genotoxic chemicals?
The cytokinesis-block micronucleus assay is a technique used ot measure the presence and the level of DNA damage. The assay contains a biomarker of chromosome breakage and/or loss, a biomarker of DNA misrepair and/or telomere end-fusions and a biomarker of elimination of amplified DNA and/or DNA repair complexes. It therefore blocks cytokinesis in cells and prevents complete division resulting in binucleated cells (BNC). This study administered the CBMA on cultured human lymphocytes after they intentionally recieved in-vivo exposure to the genotoxic mycotoxin chemical called Cytochalasin B. It was found the assays sensitivity in detecting induced MN as a result of the Cytochalasin affects is limited because of the late addition of cytochalasin B (Cyt-B) during the culture period. Therefore the BNC that are scored do not always represent cells that have the genotoxic deformity but instead are still in the process of dividing. It was also mentioned that the assay had another limitation because the genotoxic DNA damage induced could be repaired prior to the detection of the BNC.
External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.
Actin: A globular multi-functional protein that forms microfilaments.
Anillin: A scaffolding protein which interacts with RhoA, myosin II, actin and CYK-4, linking the central spindle with the equatorial cortex whilst also recruiting septin to the contractile ring.
APC: (anaphase-promoting complex) An E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome.
Aurora B-Kinase: A protein that functions in the attachment of the mitotic spindle to the centromere.
Borealin: A human protein that functions as a novel chromosomal passenger required for stability of the bipolar mitotic spindle.
CDK1: (cyclin-dependent kinase 1) A highly conserved protein that functions as a serine kinase and is a key player in cell cycle regulation.
CeMKLP1: (caenorhabditis elegans mitotic kinesin-like protein) A protein that regulates spindle midzone/midbody formation and cytokinesis in human cells.
CIT-K: (citron kinase) CIT-K is required to maintain anillin and RhoA at the midbody.
Clathrin mediated endocytosis: A process by which cells internalize molecules (endocytosis) by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized.
Cleavage furrow: A groove formed from the cell membrane in a dividing cell as the contractile ring tightens.
Clostridium botulinum C3-toxin: A toxin that causes the addition of one or more ADP-ribose moieties to Rho-like proteins.
CPC: (chromosome passenger complex) A eukaryotically conserved protein complex that localizes to kinetochores in early mitosis, the spindle mid-zone in anaphase B and to the telophase midbody.
CYK4: (cytokinesis defect family member 4) A Rho family GTPase activating protein (GAP) required for central spindle formation and cytokinesis.
ECT2: (epithelial cell transforming sequence 2 oncogene) A protein that in humans is encoded by the ECT2 gene. It acts as a transforming protein that is related to Rho-specific exchange factors and yeast cell cycle regulators.
GAP: (guanosine triphosphatase activating protein) A family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling event.
Hemicellulose: A group of complex carbohydrates that, with other carbohydrates surround the cellulose fibres of plant cells.
INCENP: (inner centromere protein) A regulatory protein in the chromosome passenger complex. It is involved in regulation of the catalytic protein Aurora B.
MAP: (mitogen-activated protein kinases) Serine/threonine-specific protein kinases involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflammatory cytokines.
Microfilaments: Linear polymers of actin subunits which are flexible and relatively strong, resisting buckling by multi-piconewton compressive forces and filament fracture by nanonewton tensile forces.
Microtubules: Fibrous, hollow rods, that function primarily to help support and shape the cell. They also function as routes along which organelles can move.
MKLP1: (mitotic kinesin-like protein 1) A kinesin protein belonging to the family of motor proteins involved in movement of intracellular materials during mitosis and cytokinesis.
Myosin II: A family of ATP-dependent motor proteins responsible for producing muscle contraction in muscle cells.
Pectin: A group of water-soluble colloidal carbohydrates of high molecular weight.
Phragmoplast: A plant cell specific structure that forms during late cytokinesis.
PRC1:(protein regulator of cytokinesis 1) A protein that in humans is encoded by the PRC1 gene that is involved in microtubule bundling and reorganisation.
RACGAP1: (Rac GTPase-activating protein 1) This protein plays a regulatory role in initiating cytokinesis, controlling cell growth and differentiation of hematopoietic cells, regulating spermatogenesis, and in neuronal proliferation.
RhoA: (Rho family of GTPases ) A small family of signaling G proteins that regulate many aspects of intracellular actin dynamics.
Schizosaccharomyces pombe: Also called "fission yeast", it is a species of yeast.
Spastin: A microtubule severing protein that disassembles microtubule bundles inside the midbody by binding to an ESCRT-III associated protein: Charged Multivesicular Body Protein 1B (CHMP1B).
Survivin: A protein that belongs to the inhibitor of apoptosis (IAP) family.
Spindle apparatus: A network of microtubules which separates chromosomes between daughter cells during cell division.
Spindle midzone: A structure composed of nonkinetochore microtubules that forms between the separating chromatin during anaphase.
ZEN-4: (zygotic lethal enclosure abnormal 4) is required for cytokinesis, localizes to the spindle midzone, and is a key component of the centralspindlin complex.
- HeLa cell in the final stage of cytokinesis.jpg
HeLa cell in the final stage of cytokinesis.
- Early Stages of Cytokinesis.jpg
A cartoon depicting cytokinesis in its early stages before it breaks off into two daughter cells. --~~~~ Image Z3293267 deleted for copyright.
- <pubmed> 19197328</pubmed>
- <pubmed>16209923 </pubmed>
- <pubmed>19000169 </pubmed>
- <pubmed>19203582 </pubmed>
- Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G