Genome mapping involves locating genes on chromosomes and determining the relative distances between genes. There are two main types of maps: genetic linkage maps which show the arrangement of genes based on their inheritance patterns, and physical maps which provide actual distances between landmarks on chromosomes. Physical maps can be further divided into cytogenetic maps, radiation hybrid maps, and sequence maps, with the complete DNA sequence being the ultimate physical map. Mapping methods include linkage analysis using genetic markers, as well as transformation, transduction, and sequencing of bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
Cell cell hybridization or somatic cell hybridizationSubhradeep sarkar
What is Cell-Cell Hybridization?
History
More about Somatic cell Hybridization
Mapping of genes by somatic cell Hybridization
Hybridoma technology
Other Applications of Somatic Cell Hybridization
Cell cell hybridization or somatic cell hybridizationSubhradeep sarkar
What is Cell-Cell Hybridization?
History
More about Somatic cell Hybridization
Mapping of genes by somatic cell Hybridization
Hybridoma technology
Other Applications of Somatic Cell Hybridization
despite of the enormous genomic diversity, the phage genome mapping is being done using a plethora of techniques,which includes both genetic mapping and physical mapping
A complete set of chromosomes/genes inherited as a unit from one parent called genome. The entire genetic complement of a living organism.
The total amount of genetic information in the chromosomes of an organism, including its genes and DNA sequences. The genome of eukaryotes is made up of a single, haploid set of chromosomes that is contained in the nucleus of every cell and exists in two copies in the chromosomes of all cells except reproductive and red blood cells. The human genome is made up of about 35,000 genes.
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
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The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
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Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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2. Genome mapping
Introduction: locating of a specific gene to
particular region of a chromosome and
determining the location of and relative distances
between genes on the chromosome.
There are two types of maps: genetic linkage map
and physical map.
3. Genome mapping
The genetic linkage map shows the arrangement
of genes and genetic markers along the
chromosomes as calculated by the frequency with
which they are inherited together.
4. Genome mapping
The physical map is representation of the
chromosomes, providing the physical distance
between landmarks on the chromosome, ideally
measured in nucleotide bases.
5. Genome mapping
Physical maps can be divided into three general
types: chromosomal or cytogenetic maps,
radiation hybrid (RH) maps, and sequence maps.
The ultimate physical map is the complete
sequence itself.
6. Law of independent assortment
Mendel: States that genes are transmitted from
parents to offspring independently of one
another.
If a person has blood group A (e.g. genotype AO) and brown eyes (e.g.
genotype Bb, where B is the allele for brown and b is the allele for
blue eyes), the AO alleles are transmitted to the offspring
independently of the Bb alleles.
However, not all genes are inherited independently of one another.
7. Linked genes
Genes that are located on the same chromosome
and are described as linked genes.
If each chromosome were to be transmitted from parent to offspring
as a whole and unaltered structure, it would be expected that all the
genes located on the same chromosome would be transmitted
together as a block and not independently of one another as proposed
in Mendel's law.
8. Recombination
However, linked genes are not always transmitted
en bloc because of the phenomenon of
recombination.
One of the fundamental events that occur in
meiosis is crossing over in which homologous
chromosomes exchange segments causing a
reshuffling of genes.
9. GENETIC DISTANCE
If genes are far apart on the same chromosome, it
is likely that recombination occurs. Conversely, if
they are very close together, they are more likely
to be transmitted as a block .
10. Frequency of recombination
The frequency of recombination of two genes is
proportional to the distance between them.
The frequency with which recombination occurs
in the offspring is expressed as a percentage.
11. Frequency of recombination
Genes which are very close together (closely
linked) will have a very small recombination
frequency (e.g. 1%).
A recombination frequency of 1% means that only
one out of 100 offspring was the combination of
two genes different from that in their parents.
12. Frequency of recombination
In contrast, genes that are very far apart on the
same chromosome or those that are on different
chromosomes are equally likely to be transmitted
together or separately and so would have a
recombinant frequency of 50%.
13. centi-Morgan
The centi-Morgan which is defined as the distance
between two genes in which recombination occurs
with a frequency of 1%.
The unit of gene distance is also called
a map unit.
15. Genetic Markers
Genes can be mapped by linkage studies with
polymorphic markers, which are nucleotide
sequences identifiable at specific sites along the
genome.
Numerous markers have been identified
throughout the genome using restriction
endonucleases and so it is possible to construct
maps of disease genes in relation to closely linked
markers.
16. Restriction endonuclease
Restriction endonucleases are naturally occurring
enzymes produced by bacteria as a defence
against invasion by viruses. The bacterial
endonucleases cut the viral DNA thus restricting
its further proliferation.
17. Restriction endonuclease
Using a large number of restriction endonucleases,
it is likely that one finds one or more RFLPs close
to the gene of interest.
Such RFLPs are then used as markers for linkage
studies with known genes. Linkage studies have
been one of the most important tools for gene
mapping.
18. Marker!!
Although the gene causing a particular trait may
not be known it is possible to identify markers
which are very closely linked to it.
19. Cotransduction
If two genes are close together along the
chromosome, a bacteriophage may package a
single piece of the chromosome that carries both
genes and transfer that piece to another
bacterium.
20. Cotransduction
In genetic mapping studies, cotransduction is used
to determine the order and distance between
genes that lie fairly close to each other.
21. Cloning vector
A cloning vector is a small piece of DNA, taken
from a virus, a plasmid, or…, that can be stably
maintained in an organism, and into which a
foreign DNA fragment can be inserted for cloning
purposes.
22. Cloning vector
The vector therefore contains features that allow
for the convenient insertion or removal of DNA
fragment in or out of the vector, for example by
treating the vector and the foreign DNA with a
restriction enzyme.
23. Types of cloning vectors
There are many types of cloning vectors, but the
most commonly-used ones are genetically
engineered plasmids.
Cloning is generally first performed using
Escherichia coli, and cloning vectors in E. coli
include plasmids, bacteriophages (such as phage
λ), cosmids, and bacterial artificial chromosomes
(BACs).
24. Types of cloning vectors
Some DNA however cannot be stably maintained
in E. coli, for example very large DNA fragment,
and other organisms such as yeast may be used.
Cloning vector in yeast include yeast artificial
chromosomes (YACs).
25. Cosmid
Cosmids are plasmids that incorporate a segment
of bacteriophage λ DNA that has the cohesive end
site (cos) which contains elements required for
packaging DNA into λ particles.
It is normally used to clone large DNA fragments
between 28 to 45 Kb.
26. Fosmid
Fosmids are similar to cosmids but are based on
the bacterial F-plasmid.
Fosmids can hold DNA inserts of up to 40 kb in
size; often the source of the insert is random
genomic DNA.
27. F’ Factors
► If
excision of F from the chromosome is not
precise, a small section of host chromosome
may be carried with the plasmid, creating an F’
(F-prime) plasmid. An F’ plasmid is named for
the gene(s) it carries, e.g., F’ (lac).
28. F’ Factors
►
F’ cells can conjugate with F- cells, and thus
introduce the bacterial gene(s) it carries. The
recipient already has a set of bacterial genes, and
so will be merodiploid (partially diploid) for those that
are introduced. This is F-duction (sometimes called
sexduction).
29. Bacterial artificial chromosome (BAC)
A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional fertility plasmid
(or F-plasmid), used for transforming and cloning
in bacteria, usually E. coli.
30. Bacterial artificial chromosome (BAC)
The bacterial artificial chromosome's usual insert
size is 150-350 kbp.
A similar cloning vector called a PAC has also been
produced from the bacterial P1-plasmid.
31. Bacterial artificial chromosome (BAC)
BACs are often used to sequence the genome of
organisms in genome projects, for example the
Human Genome Project.
A short piece of the organism's DNA is amplified as
an insert in BACs, and then sequenced. Finally,
the sequenced parts are rearranged in silico,
resulting in the genomic sequence of the
organism.
32. Common gene components of BAC
oriS, repE - F
for plasmid replication and regulation of copy number.
parA and parB
for partitioning F plasmid DNA to daughter cells during
division and ensures stable maintenance of the BAC.
A selectable marker
for antibiotic resistance; some BACs also have lacZ at the
cloning site for blue/white selection.
T7 & Sp6
phage promoters for transcription of inserted genes.
34. Bacterial artificial chromosome (BAC)
Transfection is the process of deliberately introducing
nucleic acids into cells. The term is used notably for nonviral methods in eukaryotic cells.
Microinjection refers to the process of using a glass
micropipette to insert substances at a microscopic or
borderline macroscopic level into a single living cell. It is
a simple mechanical process in which a needle roughly 0.5
to 5 micrometers in diameter penetrates the cell
membrane and/or the nuclear envelope.
35. Yeast artificial chromosome
A yeast artificial chromosome (YAC) is a vector
used to clone DNA fragments larger than 100 kb
and up to 3000 kb.
YACs are useful for the physical mapping of
complex genomes and for the cloning of large
genes.
36. Yeast artificial chromosome
A YAC is built using an initial circular plasmid,
which is typically broken into two linear
molecules using restriction enzymes; DNA ligase is
then used to ligate a sequence or gene of interest
between the two linear molecules, forming a
single large linear piece of DNA.
37. Yeast artificial chromosome advantage
Yeast expression vectors, such as YACs, YIps (yeast
integrating plasmids), and YEps (yeast episomal
plasmids), have an advantage over bacterial
artificial chromosomes (BACs) in that they can be
used to express eukaryotic proteins that require
posttranslational modification.
BUT YACs are significantly less stable than BACs.
38. Using Conjugation to Map Bacterial Genes
Conjugation experiments to map genes begin with appropriate
Hfr strains selected from the progeny of F+ X F- crosses.
41. Genetic Mapping in Bacteria by Transformation
Transformation is used to map genes in situations where
mapping by conjugation or transduction is not possible.
Donor DNA is extracted and purified, broken into fragments, and added to
a recipient strain of bacteria. Donor and recipient will have detectable
differences in phenotype, and therefore genotype.
If the DNA fragment undergoes homologous recombination with the
recipient’s chromosome, a new phenotype may be produced.
Transformants are detected by testing for phenotypic changes.
43. Transformation experiments are used to determine:
Whether
genes are linked (physically close on the bacterial
chromosome).
The
The
order of genes on the genetic map.
map distance between genes. Recombination
frequencies are used to infer map distances.
45. Transduction Mapping of Bacterial Chromosomes
Closely linked genes are cotransduced at high frequency,
allowing a detailed genetic map to be generated. For
example:
(1) Of the leu+ selected transductants, 50% have aziR and
2% have thr+.
(2) Of the thr+ selected transductants, 3% have leu+, and
0% have aziR.
(3) This gives the map order: thr--leu------azi.
46. Specialized Transduction
Specialized transduction is useful for moving specific genes
between bacteria, but not for general genetic mapping.
Some phages transduce only certain regions of the chromosome,
corresponding with their integration site(s). An example is λ in E.
coli:
i. Excision is usually precise.
ii. Rarely excision results in genetic exchange, with a fragment
ofλDNA remaining in the E. coli chromosome, and some bacterial
DNA (e.g., gal+) added to theλchromosome.
iii. The resulting transducing phage is designated λd gal+ (d for
defective, since not all phage genes are present).
iv. λd gal+ can replicate and lyse the host cell, since allλgenes are
present either on the phage or bacterial chromosome.
47. Specialized Transduction
Because transducing phage are only rarely produced, a
low- frequency transducing (LFT) lysate results. Infection
of gal bacterial cells results in two types of transductants:
i.
ii.
Unstable transductants result when wild-type
λintegrates first at its normal attλ site. λd gal+ then
integrates into the wild-typeλ, producing a double
lysogen with both types of λ integrated.
Stable transductants are produced when a cell is
infected only by a λd gal+ phage, and the gal+ allele
is recombined into the host chromosome by double
cross-over with gal.
50. Mapping Genes of Bacteriophages
1. Phage genes are mapped by 2-, 3- or 4-gene crosses,
involving bacteria infected with phages of different
genotypes.
a. Progeny phage are counted using a plaque assay in which each
phage produces a cleared area in a bacterial lawn.
b. Distinguishable phage phenotypes include mutants with
different plaque morphology. An example is strains of T2
differing in plaque morphology and/or host range.
c. The h and r genes are mapped by infecting E. coli strain B
simultaneously with two phages, h+ r and h r+.
53. Fine-Structure Analysis of a Bacteriophage
Gene
Intragenic mapping determines mutation sites within
genes.
54. Fine-Structure Analysis of a Bacteriophage
Gene
Benzer’s fine-structure mapping of phage T4
used similar experiments involving the rII gene.
a. Different rII mutations of T4 were used,
each with the characteristic large clear
plaques and limited host range.
b. T4 with the wild-type r+ gene infects E. coil
strains B and K12(λ). For rII T4, strain B is
permissive but K12(λ) is nonpermissive.
55. Recombination Analysis of rII Mutants
1.
2.
3.
Benzer’s fine-structure mapping involved 60
independently isolated rII mutants, which were
crossed in all possible combinations, using E. coli B as
the permissive host.
A linear map was constructed from the recombination
data from all crosses of the 60 rII mutants.
Later experiments have observed recombination
between adjacent base pairs, indicating that the base
pair is both the unit of mutation and the unit of
recombination. This replaced the older idea that the
gene was indivisible.
56. Benzer’s general procedure for determining the number of r+ recombinants
from a cross involving two rII mutants of T4
Reversion
To Wild tye
58. Deletion Mapping
1. Benzer eventually mapped over 3,000 rII mutants. A
deletion mapping technique was used to simplify
these studies. a. Some of the mutants did not revert, nor did they recombine
to produce r+ phage in crosses with a variety of rII mutants.
These were deletion mutants.
b. The systematic approach crossed each unknown rII mutant
with a set of seven standard deletion mutants defining the
seven main segments of the rII region.
c. Once a region for the mutation was known, the new mutant
was crossed with members of the relevant secondary set of
reference deletions. Analysis of recombination or
nonrecombination enabled more precise localization of the
mutation site
61. Defining Genes by Complementation (Cis-Trans)
Tests
1. The complementation test determines how many genes
are involved in a set of mutations that produce a given
phenotype.
2. The T4 rII region has two genes, rIIA and rIIB. A
mutation in either gene produces the rII phenotype for
both plaque morphology and host range.
62. Defining Genes by Complementation (Cis-Trans)
Tests
3. In Benzer’s work, nonpermissive strain K12(λ) was
infected with pairs of rII mutants. Neither can grow
alone in this strain.
a. If progeny are produced, the two mutants have complemented
each other by providing different gene functions, either by
genetic recombination (producing a few plaques) or
complementation (lysing the entire lawn) (Figure 14.23).
i. Infect bacterium with two phage genomes. Genotype of
one is rIIA+ rIIB, and of the other is rIIA rIIB+.
ii. One phage provides the rIIA product, the other the rIIB
product, and so the phage lytic cycle occurs.
b. If no progeny are produced, both mutations are in the same
functional unit. Both mutants produce the same defective
product (e.g., the rIIA product), and so the phage lytic cycle
cannot occur.
63. Defining Genes by Complementation (Cis-Trans)
Tests
c.Benzer’s work showed two functional units for the rII
phenotype, the complementation groups rIIA and rIIB.
Both gene products must be produced for the lytic
cycle to occur.
d. Alleles may be arranged two different ways in cistrans complementation experiments:
e. When the mutant alleles are on two different
chromosomes, as in the complementation
experiment above, they are in the trans
configuration.
66. 4. Benzer called the genetic unit of function defined by a
cis-trans complementation test a cistron. Defined as
the smallest segment of DNA encoding an RNA, cistrons
are now usually referred to as genes.
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مپینگ یعنی: تعیین محل ژنها روی کروموزوم و مشخص شدن فاصله ی بین ژنها
مپینگ بوسیله ارتباط ژنتیکی، ترتیب ژنها و مارکرهارو روی کروموزوم نشون میده و این روش برپایه فرکانس به ارث رسیدن یک ژن استوراه.
مپینگ فیزیکی، به تعیین فاصله ژنها بر اساس تعداد نوکلئوتید می پردازه.
نقشه فیزیکی به سه روش: ....و در نهایت به سکوئنسینگ ختم میشه.
قانون ترتیب مستقل:اللهایژنهایمختلفهنگامجداشدنازهمبهطور مستقل عملمیکنند.بطور مثال در فردی ک گروه خونی A داره و چشمهای قهوه ای، ژم مسئول این دو خصوصیت بطور مستقل ازهم منتقل میشن و نه وابسته به هم. ولی همه ی ژن ها هم اینچنین نیستن.
ژنهای موجود روی یک کروموزوم، ژنهای مرتبط یا linked genesگفته میشن. چون طبق نظریه مندل همه ی کروموزوم در زمان میتوز بصورت یکپارچه و بدون تغییر منتقل میشه
نوترکیبی پدیده ایه ک قانون مندلو نقض میکنه. و باعث تغییر در ترتیب ژنها میشه
در مورد ژنهایی ک کنار هم نیستن احتمال بروز نوترکیبی بیشتره. برعکس، ژنهایی که نزدیک هم هستن احتمال اینکه به صورت یک بلوک منتقل بشن بیشتره.
این شاخص متناسب با فاصله بین دو ژنه و بصورت درصد بیان میشه
ژنهای نزدیک هم فرکانس کمتری دارنمعنی فرکانس 1درصد اینه ک فقط 1درصد اولاد در ژنهای هدف بررسی دارای تفاوت هایی با والد خودشون بودن.
برعکس، ژنهایی ک دور ازهم روی یک کروموزوم و یا روی کروموزومهای متفاوت قرار دارند، با حداکثر فرکانس نوترکیبی ک مقدار آن برار با 50درصد است، روبرو هستن.
توماس مرگان، بعد از بررسی های زیاد روی نوترکیبی در خصوصیات مگس میوه، به یک مقیاس ژنتیکی دست پیدا کرد. امروزه به این واحد سنتی مرگان گفته میشه و عبارت است از فاصله دو ژن ک فرکانس نوترکیبی اونا برابر با 1درصد باشه. به سنتی مرگان، یک map unit هم گفته میشه.
به یک توالی قابل تشخیص در دی ان ای، یک مارکر ژنتیکی گفته میشه. مثل توالی های مورد هدف آنزیم های محدود کننده.
این آنزیمها در باکتری ها عمدتاً به عنوان دفاعی در برابر ژنوم ویروسها تولید میشن.
گاهی برای بدست آوردن توالی هدف لازمه از آ.م زیادی استفاده بشه. این توالی ها بعداً بعنوان مارکر هایی برای پیدا کردن رابطه بین ژنها استفاده میشه. بررسی این ارتباطات وسیله ای برای ترسیم نقشه ژنتیکی ژنومه.
اگرچه گاهی ژنهای مسئول یک خصوصیت فنوتیپی قابل تشخیص نباشن ولی با شناخت مارکر های وابسته به اون ژن میشه محل اون ژنو فهمید.
اگر دوتاژن بهم نزدیک باشن امکان اینکه توسط باکتریوفاژها بطور همزمان منتقل بشن زیاده ک به این کوترانسداکشن میگیم
در مپینگ از کوترانسداکشن برای تعیین ترتیب و فاصله ژنهای نزدیک بهم استفاده میشه.
کلون وکتور قطعه ای کوچک از دی ان ایه ک از ویروس یا پلاسمید یا سلول دیگری گرفته شده و قادره در سلول هدف دوام بیاره و در دی ان ای اون وارد بشه.
وکتور قادره به دی ان ای هدف وارد بشه و یا ازون خارج بشه. این کار ممکنه با استفاده از یک آنزیم.محدود کننده انجام بشه.
وکتورها انواع مختلف دارن ولی عمدتاً از پلاسمیدها استفاده میشه. در ابتدا از ای.کولا ی برای کلون کردن ژن استفاده شد و وکتورهای ژنها هم پلاسمید ها، باکتریوفاژها، کاسمیدها و یا از کروموزومهای باکتریایی صناعی یا بکس استفاده شد.
در مواردی ک از قطعه دی ان ای ناپایداره و در ای.کولای هضم میشه، مثل وقتی ک طول دی ان ای زیاده، از وکتور یوکاریوتی مثل کروموزوم صناعی مخمری یا یاکس استفاده میشه
کاسمیدها، پلاسمیدهایی هستن ک بخشی از باکتریوفاژ لاندارو در خودشون دارن. این بخش دارای توالی cos هستش و باعث بسته بندی شدن دی ان ای در کپسید فاژ لاندا میشه. از کاسمیدها عمدتاً برای کلون کردن قطعات بیشتر از 28 تا 45kb استفاده میشه.
فاسمیدها بر پایه پلاسمید اف در باکتری ها ساخته میشن. و قادره دی ان ای تا 40kb منتقل کنه.
اگه خروج فاکتور اف از کروموزوم دقیق نباشه به اون اف پریم میگیم. در شکل چون ژن لک همراه اف پریمه به اون اف پریم لک گفته میشه.
اف پریم میتونه با اف منفی ها کانژوگه بشه و در این صورت میزبان به صورت مروپلوئید درمیاد. به این روش انتقال ژن، اف داکشن یا سکس داکشن هم گفته میشه.
بک ها بر پایه پلاسمی اف ساخته میشن و برای ترنسفورم و کلون ژن در باکتری ها، خصوصاً ای.کولای استفاده میشن.
بک قادره از 150 تا 350kb رو منتقل کنه.پک هم نوع دیگه وکتوره ک بر پایه پلاسمید پی وان سخته شده
از بک معمولاً در پروژه های سکانس ژنوم اورگانیزم ها استفاده میشه، مثل پروژه ژنوم انسان.یک قطعه کوچیک دی ان ای اورگانیزم بعد ورود به یک بک، کلون میشه و سپس سکانس میشه. سپس قسمت های سکانس شده توسط نرم افزار کامپیوتری به هم مرتبط میشن و توالی ژنوم اورگانیزم مشخص میشه.
ترانسفکشن به انتقال اسید نوکلئیک به میزبان با استفاده از وکتورهای غیر ویروسی گفته میشه. میکرواینجکشن هم ب انتقال مواد با استفاده از میکروپیپت به داخل سلول زنده گفته میشه. این میکرو پیپت ها از 0.5 تا 5 میکرومتر قطر دارن. و حتی میتونن موادو مستقیم به هسته منتقل کنن.
یاک هم کروموزوم ساختگی مخمریه ک دی ان ای 100 تا 3000kb رو منتقل میکنه. و برای کلون ژن های بزرگ استفاده میشه.
یاک در واقع یک پلاسمیده ک توسط آنزیم محدود کننده بصورت دو قطعه خطی در میاد. ژن مورد نظر توسط آنزیم دی ان ای لیگاز به دو قطعه متصل میشه و کلاً تشکیل یک دی ان ای خطی میدن.
وکتور های مخمری مثل یاک ، وای ال پی ،و وای ای پی مزیت هایی نسبت به بک دارن و اون اینه ک از اینها میشه برای بررسی پروتئین هایی ک ب تغییرات پس از ترجمه نیاز دارن، در یوکاردوت ها استفاده کرد. ولی نسبت به بک از پایداری کمتری برخوردارن.
تعیین نقشه ژنتیکی با استفاده از کانژوگاسیون، با استفاده از سویه های اچ اف آر حاصل از ترکیب اف + و اف – شروع شد.
زمان انتقال اولین ژن، زمان 0 در نظر گرفته میشه و به ترتیب....زمان انتقال برای هر ژن، در صورت تکرار آزمایش، مشابه زمان قبله و این نشون میده ک محل این ژنها روی کروموزومه.از محیط انتخابی برای آنالیز ترانسکانژوگانت ها استفاده میشه.
در هر سویه اچ اف آر، کروموزوم دارای یک پلاسمید اف هست ک با توجه ب سویه، جهت گیری و محل آریجین اون متفاوته. با در نظر گرفتن نواحی اورلپ در توالی های ترنسفر شده در طول کانژوگاسیون، کروی بودن ژنوم پروکاریوت اثبات میشه.
ترانسفورماسیون وقتی انجام میشه ک انجام ترانسداکشن و کانژوگاسیون مقدور نباشه. برخی باکتری ها مثل باسیلوس سرئوس بدون انجام هیچ تریتی توانایی دریافت دی ان ای دارن ولی برخی دیگه فقط تحت شریط خاصی قادرن دی ان ای خارجیو دریافت کنن. دی ان ای هدف بعد از تخلیص، خورد میشه و به محیطی ک باکتری درون وجود داره اضافه میشه. اورگانیزم هدف و باکتری میزبان دارای تفاوتهای فنوتیپی هستن و در نتیجه از لحاظ ژنوتیپی هم باهم متفاوتن. اگر نوترکیبی انجام بشه باتوجه به اینکه باعث بروز تغییر در خصوصیات فنوتیپی میشه، پس قابل تشخیصه.
ترانسفورماسیون کامل فقط در تعداد کمی از سلول ها اتفاق میوفته.پس از تکثیر نهایی دو ژنوتیپ بوجود میاد ک از طریق تفاوتهای فنوتیپی قابل تشخیصن.
ترانسفورماسین به سه منظور انجام میشه: یکی اینکه آیا ژنها به هم متصل هستن یا نه، دوم اینکه ترتیب اونا چطوره و سوم اینکه فاصله بین ژنها چقدره.
دی ان ای های کوچکتر ک فقط دارای چند ژن هستن بهتر ترانسفورم میشنو انتقال همزمان دو ژن نشون میده ک این ژنها به هم نزدیک هستن. درشکل دو ژن پی و پیو کنار هم هستن. پس همیشه با هم ترانسفورم میشن. اگر گاهی اوقات ژن او با کیو ترانسفورم بشه اونوقت باید ترتیب ژنهارو بر اساس مقایسه فرکانس نوترکیبی دوتا ژن محاسبه کرد.
اینجا هم انتقال همزمان چند ژن و بررسی فرکانس نوترکیبی اونها در مقایسه باهم، زمینه تعیین دقیق نقشه ژنتیکی باکتری رو فراهم میکنه.
برخی فاژها فقط نواحی خاصی از ژنومو ترنسدیوس می کنن. مثل فاژ لاندا ک در محل attLanda وارد میشه. این توالی بین ژنهای بیو و گال قرار داره. و پروفاژ اون توسط یک پر.تئین فاژ رپرسور حفظ میشه. اگر پروفاژ تحریک بشه توسط کراس اور خارج میشه. خروجش معمولاً دقیقه ولی گاهی بخشی از دی ان ای ای.کولایو هم خارج میکنه. چون این فاژ همه ی ژنوم فاژو نداره به اون فاژ دیفکتیو یا ناقص گفته میشه. این فاژ برای تکثیر خودش وابسته به فاژهای سالمه.
چون تکثیر این فاژ بخاطر وابستگیش ب فاژهای سالم، کمه، کمتر باعث لیز میزبان میشه. سویه های سالم در محل نرمال ورود وارد میشن. بعد فاژهای ناقص وارد توالی فاژهای ناقص می شن. و میزبان دابل لایزوژن میشه. این میزبان هتروزایگوته و قادره گالاکتوزو تخمیر کنه. در اثر تحریک سویه کامل، اون میتونه به لایتیک تبدیل بشه. وقتیکه میزبان با سویه ناقص آلوده بشه و ژن گال هم بصورت دابل کراس اور وارد ژنوم میزبان بشه، این انتقال پاداره.
مپینگ ژنوم باکتریو فاژ ها توسط 2 تا 4 ژن انجام میشه و باکتری رو با چندین ژنوتیپ فاژ آلوده میکنن. تشخیص تغییرات ژنوتیپی برپایه تغییرات ظاهری پلاک هاست. ژنهای اچ و آر توسط ای.کولای سویه بی ک با هر دو ژنوتیپ .... و ... آلوده شده صورت میگیرد.
h+ lyses ONLY E. coli strain Br is a mutant producing large distinct plaques).h lyses both B and B/2 E. coli, r+ produces the wild-type small plaque with fuzzy borders).
مپینگ داخل ژنی ک به تشخیص جهش های داخل یک ژن میپردازه.
برای بررسی ژن آرتوو استفاده شده. فاژهای تی فور با موتاسیونهای متفاوت در ناحیه آرتوو و با خصوصیات توانایی تولید پلاک بزرگ استفاده شدن. ژنوتیپ r+ توانایی آلوده کردن strains B and K12(λ). ای.کولای را داره و زنوتیپ r|| برای فاژ K12(λ) مجاز نیست.
این دانشمند 60 ایزوله مستقل آر توو رو جداسازی کرد ک همه ی حالت های موتاسیون در این ژنو پوشش میدن. یک نقشه خطی از اطلاعات حاصل از نوترکیبی تهیه کرد. و در مطالعات بعدی هم نوترکیبی بازهای مجاورو بررسی کرد و نشان داد ک جفت بازها واحدهای موتاسیون و نوترکیبی هستن.
بیشترین فرکانس نوترکیبی بین سویه های آرتوو 50درصد بوده. کمترین فرکانس نوترکیبی 0.01% بوده ک باتوجه به طول ژنوم این فاصله برابر با 3 bp بوده.
در نهایت موفق شد 3000 موتانت آرتوو را تفکیک کند. برای یسته بندی راحت تر این داده ها از مپینگ حذفی استفاده کرد. برخی موتانتها در کراس شدن با سویه های دیگر به حالت اولیه برنمی گشتند. اینها موتانهای حذفی بودند. این دانشمند از بین موتانهای حذفی را به 7دسته اصلی تقسیم کرد. و سایر موتانها را مرحله به مرحله بر اساس مقایسه با این موتانهای حذفی تفکیک کرد.
Benzer’s work with > 3,000 mutants divided the rIIregion into > 300 mutable sites separable by recombination. Certain sites in the region (hot spots) have high rates of independent point mutations.
تعیین ژن با استفاده از تست های کامل سازیاین تستها مشخص می کنن ک چطور تعدادی از ژنهای دخیل در موتاسیون، در تغییرات فنوتیپی نقش دارنژن آرتوو در فاژ تی فور دارای دو ناحیه A و B است. موتاسیون در هر یک ازین دوژن باعث تر مورفولوژی پلاک می شود.
دراین آزمایش هر دو سویه در K12(λ) ای.کولای کشت داده شدن و هیچکدام قادر به رشد روی این محیطها نبودند. نسل اول نقص های خود را باتکمیل فعالیت یکدیگ را کامل کردند و یا حتی ممکن است نوترکیبی رخ دهد. اگر پلاکی دیده نشود معنی آن اینست ک هردو ژن روی یک ناحیه هستند.
این دانشمند نشان داد ک دو ناحیه عملکردی روی آرتوو وجود دارد ک فعالیت هر دو برای اجرای چرخه لایتیک لازمه. موتاسیونهای نقطه ای یا موتاسیونهای حذفی، اثری مشابه روی تست تکمیلی داشت. اگر آلل ها روی نواحی عملکردی متفاوت باشن در حالت ترانس قرار دارن و برعکس.
i. The fine-structure map indicates the boundaries of rIIA and rIIB. ii. Point mutants and deletion mutants in both rIIA and rIIB give the same results in complementation tests. iii. Deletions that span rIIA and rIIB do not complement either rIIA or rIIB.
اودر ادامه سیسترون را به عنوان واحد عملکردی دی ان ای ک یک آر ان ای کامل را کد میکند، معرفی کرد. و میشود سیسترون ها را به عنوان ژن هم در نطر گرفت.