Seminario biología molecular
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This document discusses a study investigating telomerase activation as a repair response to radiation-induced DNA damage in Y79 retinoblastoma cells. The study aims to examine the molecular mechanism of IR-induced telomerase activation in these cells. The authors cultured Y79 cells and exposed them to different doses of ionizing radiation. They found that telomerase activity increased 12-24 hours after low IR doses (<10 Gy) but decreased after higher doses (>10 Gy). IR also activated MAPK signaling pathways and increased hTERT expression levels involved in telomerase activation and DNA damage response. The conclusions were that telomerase activation is essential for repairing damaged DNA ends after IR exposure.
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Genetic code2
The document discusses genetic code, gene expression, and DNA fingerprinting. It first explains that the genetic code is the relationship between mRNA nucleotide sequences and the amino acid sequences in polypeptides. It then notes that in 1954, George Gamow proposed that the genetic code should be made up of three nucleotides to code for all 20 amino acids. Finally, it outlines the three stages of protein synthesis - initiation, elongation, and termination - and provides details on the initiation stage in prokaryotes, including the components required such as the ribosome, mRNA, initiation tRNA, and three initiation factors.
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anti sense RNA technology is one the method for gene silencing. it found very effective making Flavr savr tomato. hope this ppt will help you.
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Genetic code
This document summarizes key aspects of protein synthesis and the genetic code. It begins by explaining that protein synthesis, also called translation, uses the genetic code to translate mRNA sequences into amino acid sequences to form proteins. The genetic code is described as a dictionary that uses triplet codons to specify which of 20 amino acids will be incorporated into a growing polypeptide chain. Each codon is recognized by a complementary anticodon on transfer RNA molecules. The genetic code has several notable features, including being universal across all living things, having some redundancy with multiple codons specifying the same amino acid, and including three non-overlapping stop codons that terminate protein synthesis.
The-Genetic-code-converted.pptx
The genetic code is the set of rules by which genetic material like DNA and mRNA are translated into amino acid sequences that make up proteins. The genetic code specifies which amino acid will be added next during protein synthesis using sequences of three nucleotides called codons. While some codons specify the same amino acid, meaning the code is degenerate, this allows more codons to exist without increasing the number of amino acids used. Efforts to understand the genetic code increased after the discovery of DNA's structure, with scientists like Gamow theorizing it must use three nucleotides to code for the 20 standard amino acids.
Molecular mechanism of antisense molecules
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This document summarizes key aspects of protein synthesis and the genetic code. It begins by explaining that protein synthesis, also called translation, uses the genetic code to translate mRNA sequences into amino acid sequences to form proteins. The genetic code is described as a dictionary that uses triplet codons to specify which of 20 amino acids will be incorporated into a growing polypeptide chain. Each codon is recognized by a complementary anticodon on transfer RNA molecules. The genetic code has several notable features, including being universal across all living things, having some redundancy with multiple codons specifying the same amino acid, and including three non-overlapping stop codons that terminate protein synthesis.
The-Genetic-code-converted.pptx
The genetic code is the set of rules by which genetic material like DNA and mRNA are translated into amino acid sequences that make up proteins. The genetic code specifies which amino acid will be added next during protein synthesis using sequences of three nucleotides called codons. While some codons specify the same amino acid, meaning the code is degenerate, this allows more codons to exist without increasing the number of amino acids used. Efforts to understand the genetic code increased after the discovery of DNA's structure, with scientists like Gamow theorizing it must use three nucleotides to code for the 20 standard amino acids.
Molecular mechanism of antisense molecules
Antisense RNA is a single-stranded RNA that is complementary to messenger RNA (mRNA) and inhibits its translation. Historically, antisense RNA effects were confused with RNA interference, which involves small interfering RNAs targeting mRNA for degradation. Antisense RNA works by forming duplexes with mRNA, blocking the ribosome from accessing nucleotides or leading to degradation. Examples include transgenic Flavr Savr tomatoes with reduced ethylene production using antisense RNA against an ethylene-producing enzyme, and natural antisense RNA in mice and humans that blocks insulin-like growth factor 2 receptor mRNA. While promising, antisense RNA drugs still face challenges in design, activity, and delivery that have limited
Transgene silencing
Gene silencing, also known as RNA interference, is a natural process in plants that evolved as a defense mechanism against viruses. Transgene silencing occurs when introduced transgenes are not expressed due to this silencing process. The first evidence of this was discovered in 1990 by R. Jorgensen in petunia plants, where both an introduced gene and endogenous gene were silenced. Gene silencing can occur at the transcriptional or post-transcriptional level through mechanisms like siRNA and microRNA production. Virus-induced gene silencing is a technique used to study gene function and develop virus-resistant plants by suppressing viral gene expression. Applications of gene silencing include developing disease-resistant crops and modifying plant traits.
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Antisense RNA is a single-stranded RNA that is complementary to messenger RNA and can inhibit its translation by base pairing and physically obstructing translation machinery. An example is the hok/sok system in E. coli where the sok RNA antitoxin inhibits the hok toxin RNA. Antisense RNA has roles such as inactivating genes for polygalacturonase and ethylene synthesis. It also has potential applications in gene therapy and developing therapies for viruses and conditions like high blood pressure.
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MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
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Alkaline Phosphatase
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Terminal deoxyneucleotidyl transferase
Nucleases
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Bal31 Exonuclease III
Endonuclease
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Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
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Categorization of enzymes
Isoschizomers
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Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
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2) Telomerase is an enzyme that can rebuild telomeres and counteract their shortening. It is active in stem cells and cancer cells but not most somatic cells. Telomerase expression allows cancer cells to divide indefinitely by maintaining telomere length.
3) The document examines telomere length and telomerase activity in different plant parts and telomerase-deficient Arabidopsis mutants. It finds that telomerase knockout leads to progressive telomere shortening and chromosome f
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Transgene silencing
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Antisense rna
Antisense RNA is a single-stranded RNA that is complementary to a messenger RNA strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. Antisense RNA has long been thought of as a promising technique for disease therapy, but generally still lacks effective design, biological activity, and efficient routes of administration. Transcription of long antisense non-coding RNAs is a common phenomenon in the mammalian transcriptome and can result in transcriptional gene silencing through histone modifications and creating a heterochromatin environment around the gene. Antisense RNA plays a pivotal role in inhibiting cell translation by producing a
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Antisense technology uses short DNA sequences called oligonucleotides that are complementary to messenger RNA (mRNA) to prevent specific proteins from being synthesized. When introduced into cells, these antisense oligonucleotides bind to their target mRNA through Watson-Crick base pairing, forming RNA-DNA hybrids that are degraded by RNase H enzyme. This prevents translation and expression of the target protein. There are three generations of antisense oligonucleotides that have been developed with improved stability and targeting capabilities, including phosphorothioate, 2'-O-methyl RNA, and locked nucleic acid chemistries. Antisense technology has potential applications in treating diseases like cancer, viral infections, and genetic disorders.
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Gene silencing
Gene silencing is a mechanism of gene regulation that switches off genes without genetic modification. It can occur at the transcriptional or post-transcriptional level. Post-transcriptional gene silencing is achieved through antisense technology or RNA interference (RNAi). Antisense technology uses synthetic nucleic acid molecules that are complementary to mRNA to block translation into proteins. RNAi involves introducing double-stranded RNA that is processed into small interfering RNAs (siRNAs) that guide the RNA-induced silencing complex (RISC) to degrade mRNAs with complementary sequences, thereby silencing genes. Both antisense technology and RNAi have applications for treating diseases and studying gene function and regulation.
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The presentation discusses telomeres and telomerase in the context of cancer. It begins by defining telomeres as repetitive DNA sequences that cap the ends of chromosomes and shorten each time a cell divides. Telomerase is an enzyme that adds telomeric repeats and prevents shortening. In cancer cells, telomerase remains active and prevents telomere shortening, allowing unlimited cell division. Measuring telomerase may help detect and treat cancer by inducing senescence, though blocking it could also impair normal cell functions. The conclusion is that while inhibiting telomerase shows promise against cancer, more research is needed to minimize side effects.
Dna methylation christopher-mendoza
The document describes the process of bisulfite genomic sequencing used to analyze DNA methylation at single nucleotide resolution. Key steps include:
1) Treating DNA with sodium bisulfite to convert unmethylated cytosines to uracils while leaving methylated cytosines unchanged.
2) Amplifying target regions using PCR and primers specific to bisulfite-converted DNA.
3) Cloning and sequencing amplified regions to determine cytosine/uracil content and map methylation sites.
The multi-step process allows for high-resolution analysis of DNA methylation but new techniques may provide alternatives.
DNA Footprinting
Hello There,
DNA Footprinting Is A Molecular Biology Technique With Wide Applications In Many Areas Of Biological Sciences And Importantly It Is Used For Crime Detection In Forensic Sciences. In This Presentation, You Will Learn What It Is, The Technology, Protocol, Pictorial Representation, Applications And References For Further Study.
Antesense rna
Antisense RNA is a single-stranded RNA that is complementary to messenger RNA and can inhibit its translation by base pairing and physically obstructing translation machinery. An example is the hok/sok system in E. coli where the sok RNA antitoxin inhibits the hok toxin RNA. Antisense RNA has roles such as inactivating genes for polygalacturonase and ethylene synthesis. It also has potential applications in gene therapy and developing therapies for viruses and conditions like high blood pressure.
Dna modifying enzymes
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Oligonucleotide
Oligonucleotides therapeutics work by altering gene expression through complementary binding to mRNA. There are three major classes: antisense oligonucleotides inhibit protein expression through hybridization to mRNA, siRNA induce mRNA degradation, and miRNA inhibit translation. Oligonucleotides are modified through conjugation or encapsulation to improve delivery and stability, and resist nuclease degradation. Antisense oligonucleotides specifically designed to hybridize with mRNA can function through RNase H degradation or steric blocking of translation.
Biotechnology- Antisense Technology
This document provides an overview of antisense technology for inhibiting gene expression. It discusses how antisense oligonucleotides are complementary to mRNA and can bind to prevent translation. The mechanisms of hybridization arrest and RNaseH activation are described. Different approaches using antisense oligonucleotides that do and do not activate RNaseH are covered. Applications in agriculture and medicine are mentioned, including the development of the Flavr Savr tomato that used antisense technology to delay fruit ripening. The role of antisense oligonucleotides in drug discovery is also briefly discussed.
Restriction enzyme
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
ANTISENSE OLIGONUCLEOTIDES
This document discusses antisense oligonucleotides, which are single-stranded RNA molecules that regulate gene expression by binding to mRNA. Antisense oligonucleotides have been developed as therapeutics to treat conditions like cytomegalovirus retinitis, hypercholesterolemia, and muscular dystrophy. They work either by recruiting RNase H to degrade mRNA or by physically blocking translation. Delivery of antisense oligonucleotides into cells occurs through endocytosis or fluid-phase pinocytosis. Antisense technologies can be used in drug discovery to specifically manipulate gene expression and identify novel drug targets.
Restriction enzymes
Restriction enzymes are molecular scissors found in bacteria that cut DNA molecules at specific recognition sequences. They serve as a defensive mechanism for bacteria against bacteriophages by cleaving the phage DNA. There are over 3000 known restriction enzymes that are classified into four main types based on their composition, cofactors, and cutting mechanisms. Restriction enzymes are important tools in biotechnology for manipulating DNA sequences through cutting DNA fragments with specific sticky or blunt ends, which can then be recombined through techniques like cloning.
Antisense therapy
Antisense therapy is a form of genetic treatment that uses artificially synthesized nucleic acid sequences to bind to messenger RNA (mRNA) produced by a disease-causing gene, inactivating it and effectively turning the gene "off". This approach is being researched as a treatment for various diseases including cancers, diabetes, ALS, and muscular dystrophy. Antisense drugs work by forming DNA/RNA hybrids that are degraded by the RNase H enzyme, preventing translation of the mRNA into protein.
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Regulation of gene expression.
Ligand-gated channel.
Ligand regulated trans-membrane enzyme
Lecture 3 Early Embryogenesis and Cell Fate How is blas.pdf
Lecture 3 Early Embryogenesis and Cell Fate
How is blastopore formed? What organ will blastopores become?
The results of gastrulation: three germ layers and archenteron and blastopore
Tissues and organs from each germ layer
Procedure of neurulation and the functions and origins of each part including notochord Epithelial
cells and mesenchymal cells
Fate mapping: how to make a fate map?
Vital dye staining experiment
Fluorescent dye experiment
Genetic labeling experiment
Lecture 4 Genomic Equivalence
Germ plasm theory by August Weismann
What is genomic equivalence?
Evidence for genomic equivalence
Rouxs experiment
Cell transplant and cell removal experiment in frog embryo
Drieschs experiment
Hans Spemanns experiment
Nuclear transfer technique and animal cloning
Briggs and Kings frog cloning using blastula cell nucleus
Gurdons Xenopus cloning using intestinal cell nucleus
Ian Wilmuts sheep cloning using breast cell nucleus
Jaenischs mouse cloning using lymphocyte nucleus
The article, Nuclear Transfer Bringing in the clones
Every somatic cell has the same DNA (genome), then, how do they differentiate into different
types of cells?
Lecture 5 Genes and Transcriptional Regulation
What is the gene?
What is the genome?
Where are the genes written? (Which molecule has genes on it?)
How do genes work? What is the function of genes?
What is gene expression?
What determines the identity and function of a protein?
What determines the structure (shape) of a protein?
What determines the amino acid sequence of a protein?
Overview of gene expression
mRNA transcription
Protein translation
What is proteome?
Different types of cells have different proteome.
How different types of cells have different set of proteins although they have the same genome?
What are the housekeeping genes?
Overview of gene expression regulation
The structure of genes: Transcription initiation site, 5 UTR, exons, and introns, 3 UTR
Regulatory elements on DNA: promoters and enhancers
Transcriptional regulation
The switch on DNA for transcription regulation: enhancers and promoter
Promoter, TATA box, general transcription factors, and RNA polymerase II
Enhancers and specific transcription factors
The structure of transcription factors
How do enhancers and specific transcription initiate transcription?
Chromatin structure and transcription regulation
Nucleosomes, histone octamer and chromatin structure
Histone acetylation and histone methylation
Histone structure modifying enzymes: histone methyltransferase, histone demethylase, histone
acetyltransferase, and histone deacetylase
Heterochromatin vs. euchromatin
Steps of the initiation of transcription
Enhancer modularity
How can one gene be transcribed in multiple types of cells?
One gene can have multiple enhancers
How can multiple genes be transcribed in one type of cell?
A cell has a variety of specific transcription factors that bind to different enhancers of different
genes.
Multiple genes can have the same enhancers for the same speci.
Telomers and Telomerases
Telomeres are repetitive DNA sequences at the ends of chromosomes that protect them from deterioration during cell division. Telomeres shorten each time a cell divides, acting as a biological clock. When telomeres become too short, cells can no longer divide and become senescent. Telomerase is an enzyme that adds telomeric repeats to chromosome ends, counteracting shortening and allowing cells to avoid senescence. Cancer cells express high levels of telomerase, allowing them to divide indefinitely. Drugs are being developed to inhibit telomerase as a cancer treatment, though they can also damage telomeres in normal cells. Future research aims to specifically target the telomerase expression in cancer cells.
Telomeres kiran br
1) Telomeres are protective nucleoprotein structures at the ends of chromosomes that shorten with each cell division due to the end replication problem.
2) Telomerase is an enzyme that adds telomeric repeats to chromosome ends to counteract shortening and allow indefinite cell division.
3) Telomerase activation allows cancer cells to avoid replicative senescence and become immortalized, while preceding telomere shortening provides a mechanism for genetic instability and tumor progression.
Telomeres kiran br
1) Telomeres are nucleoprotein structures at the ends of chromosomes that protect them from degradation. Telomerase is an enzyme that elongates telomeres by adding DNA repeats.
2) Telomerase activity is observed in over 80% of cancers and helps cancer cells proliferate indefinitely by maintaining telomere length.
3) In normal cells, telomeres shorten with each cell division due to the end-replication problem until cells enter senescence. Rare cells can reactivate telomerase to immortalize.
Epigenetics : overview and concepts
epigenetic mechanism and inheritance with little bit of effect of heavy metals and nutrition. Not very detailed but just a few key points.
CRISPR cas9 mediated TERT disruption in cancer cells
CRISPR/Cas9 was used to disrupt the TERT gene in cancer cells. Three gRNAs targeted exon regions of the TERT gene in HELA, PANC1, and SKBR3 cells, achieving up to 85% editing efficiency as determined by PCR and T7 endonuclease assays. Xenograft experiments found that editing TERT prevented tumor formation in nude mice. While effective, delivery methods need improvement and off-target effects require validation before clinical use. Future work aims to identify additional tumor-specific genes that could be targeted with CRISPR to develop new cancer therapies.
Cancer
This document discusses various aspects of carcinogenesis. It begins by describing the key properties of cancer cells such as uncontrolled growth, invasion, and metastasis. It then discusses various causes of cancer including physical, chemical, and biological agents. Radiation, chemicals, and viruses can all act as carcinogens. The document goes on to describe how carcinogens interact with and damage DNA. It discusses initiation and promotion in carcinogenesis and the roles of oncogenes and tumor suppressor genes such as p53. The document also covers topics like telomerase, metastasis, and the tendency of tumors to progress in malignancy.
Genetic control
This document discusses genetic control and regulation at the molecular level. It covers DNA and RNA structure, transcription, translation, gene regulation, and cell division. The central dogma of molecular genetics is described involving DNA transcription to RNA and translation to protein. The types of RNA are defined including mRNA, tRNA, rRNA. Transcription and translation processes are explained in detail. Gene regulation mechanisms like promoters, transcription factors, and feedback systems are covered. Finally, the document discusses cell reproduction through DNA replication and cell mitosis.
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Seminario biología molecular
- 1. Alejandra Mejía Mora Daniela Martínez Laverde 3º Semestre medicina U.P.B
- 2. Texto adicional Texto adicional Texto adicional texto adicional Texto adicional texto adicional Masaharu Akiyama, Kohji Ozaki, Takeshi Kawano, Osamu Yamada Kiyotaka Kawauchi, Hiroyuki Ida, Hisashi Yamada. TELOMERASE ACTIVATION AS ATELOMERASE ACTIVATION AS A REPAIR RESPONSE TO RADIATION-REPAIR RESPONSE TO RADIATION- INDUCED DNA DAMAGE IN Y79INDUCED DNA DAMAGE IN Y79 RETINOBLASTOMA CELLSRETINOBLASTOMA CELLS
- 3. INTRODUCTION
- 5. Ribonucleoprotein enzyme made of protein and RNA subunits. Found in fetal tissues, adult germ cells, and also tumor cells. Elongates chromosomes by adding TTAGGG sequences. Has a very low activity in somatic cells, and high in “immortal cell. Cellular aging It is due to the shortening of chromosomal telomeres. TelomeraseTelomerase
- 6. •Reverse transcriptase protein (TERT) - Essential telomerase N-terminal domain (TEN). - Telomerase RNA-binding domain (TRBD). - Reverse transcriptase domain (RT). - C-terminal extension (CTE). • RNA component (TR). • Dyskerin protein complex (blue). •Telomeric DNA. TelomeraseTelomerase
- 12. • Cancer cells are a type of malignant cell that multiply until they form a tumor that grows uncontrollably. • Detected in human cancer cells and is found to be 10-20 times more active than in normal body cells. • Telomerase activity off/on. Telomerase and cancerTelomerase and cancer
- 14. General objective • Investigate the molecular mechanism of IR-induced telomerase activation in human retinoblastoma Y79 cells.
- 15. Materiales y métodosMateriales y métodos Cultivo y reactivos Citometría de flujo Ensayo de la telomerasa Transcriptasa inversa - PCR Immunoblot
- 16. RPMI 1640 Células retinoblastoma Y79 Suero fetal bovino (10%) • L-glutamina • Penicilina • Estreptomicina 5% CO2 100% humedad 1 2 3 Wortmanina Ácido okadaico Cultivos y reactivosCultivos y reactivos
- 17. Análisis del ciclo célular FACSCalibur Citometría de flujoCitometría de flujo
- 18. Análisis de las células apoptóticas Citometría de flujoCitometría de flujo
- 19. Ensayo de telomerasaEnsayo de telomerasa
- 23. Efectos del IR en la actividad telomerasa y expresión hTERT Resultados
- 24. Efectos IR en actividad de telomerasa vs tiempo de exposición Resultados
- 25. Expresión de hTERT (RNAm) Resultados
- 26. Efecto de IR en el ciclo célular 2 Gy Resultados
- 27. 5 Gy Resultados
- 28. Resultados 10 Gy
- 29. Resultados Efecto IR sobre la apoptosis
- 30. Resultados Efecto de IR en moléculas de respuesta al daño de DNA
- 31. Resultados Efectos de la IR en la expresión de la proteína hTERT
- 32. Resultados Efectos producidos por la Wortmanina
- 33. Efectos producidos por el ácido ocadaico Resultados
- 34. Discussion REFERENCE WHAT THEY SAID AGREE OR DESAGREE M. Natarajan, S. Mohan, R. Konopinski, F. Leteurtre, X. Li, E. Gluckman, E.D. Carosella, E. Fenig, E. Beery, S. Lichtenberg, Y. Nordenberg, M. “Telomerase activity is increased 12– 24 h after a IR dose of less than 10 Gy but is decreased only after a dose greater than 10 Gy”. Agree Y. Yan, P.T. Cao, P.M. Greer, E.S. Nagengast, R.H. Kolb, M.C. Mumby, K.H. Cowan. “The G2/M checkpoint prevents cells from initiating mitosis when they sustain DNA damage during G2 or when they enter the G2 phase with unrepaired DNA damages sustained during previous S or G1 phases”. Agree R.A. Tamakawa, H.B. Fleisig, J.M.Y. Wong. “Proteins of the MAPK family, such as p38, JNK, and ERK, are also activated by IR in response to genotoxic stress”. Agree S.S. Kang, T. Kwon, D.Y. Kwon, S.I, J. Haendeler, J. Hoffmann, S. Rahman, A.M. Zeiher, S. Dimmeler. “Okadaic acid, a PP2A inhibitor, is known to activate Akt through its phosphorylation, resulting in increased telomerase activity”. Agree
- 35. Conclusions • Ionizing radiation (IR) causes DNA damage, which generates multiple processes inside the cell. • It is very important to begin any treatment for retinoblastoma avoiding the appearance of secondary cancers.
- 36. Conclusions • In studies related to DNA damage is important to analyze the cell cycle and the percentage of apoptotic cells. • Telomerase activation is essential for repairing DNA extremes damaged during the IR
- 39. Bibliography • MARTINEZ SÁNCHEZ, Lina María. Biología molecular. 7 ed. Medellín: UPB. Fac. de Medicina, 2013. 47-51, 85- 88 p. • Masaharu Akiyama, Kohji Ozaki, Takeshi Kawano, Osamu Yamada Kiyotaka Kawauchi, Hiroyuki Ida, Hisashi Yamada. “Telomerase activation as a repair response to radiation-induced DNA damage in y79 retinoblastoma cells”. 5 July 2013.
Editor's Notes
- -
- RPMI Media 1640 has a wide range of applications for mammalian cells, including the culture of fresh human lymphocytes, fusion protocols, and growth of hybrid cells.