What does T and the numbers mean in T coliphages?

What does T and the numbers mean in T coliphages?

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I have searched a number of websites and articles relating to nomenclatures or phages but never found anything explaining what T in T-4,5,6… Phages mean in the Coliphages. Also, what does the number mean? I have seen that many times, the T - even are grouped as one, why is it so? Thank you.

The "T" stands for "Type" as in type 1, type 2 etc. To quote Wikipedia on Escherichia virus T4 (emphasis mine):

Bacteriophages were first discovered by the English scientist Frederick Twort in 1915 and Félix d'Hérelle in 1917. In the late 1930s, T. L. Rakieten proposed either a mixture of raw sewerage or a lysate from E. coli infected with raw sewerage to the two researchers Milislav Demerec and Ugo Fano. These two researchers isolated T3, T4, T5, and T6 from E.coli. Also, in 1932, the researcher J. Bronfenbrenner had studied and worked on the T2 phage, at which the T2 phage was isolated from the virus.[39] This isolation was made from a fecal material rather than from sewerage. At any rate, Max Delbrück was involved in the discovery of the T even phages. His part was naming the bacteriophages into Type 1(T1), Type 2 (T2), Type 3 (T3), etc.

It turns out Wikipedia is incorrect: The original use of Type as a designation for these viruses comes from a paper published in 1945 (PDF) by M Demeric and U Fano: BACTERIOPHAGE-RESISTANT MUTANTS IN ESCHERICHIA COLI Genetics 30:119. In this paper they collected phages from Luria (of LB/Luria Broth (actually properly known as lysogeny broth)) and Delbruck amongst others and collated them all for comparison. Delbruck references them in his review article (PDF, possible paywall, summary here), also from 1945.

I could find no evidence for why the phages were numbered as they were, though strains from Luria (actually previously from his supervisor as P28 and PC) were labeled T1 (Luria alpha) and T2 (Luria gamma), so it is possible that it was simply in order that they were received or possibly in order of discovery amongst the several groups working on these viruses.

The T-even phages were grouped because of structural similarities, and are now strains of the species escherichia virus T4 in the genus Tequatrovirus (see link for taxonomy).

  • Virulence is the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism is determined by its virulence factors.
  • A key difference between the lytic and lysogenic phage cycles is that in the lytic phage, the viral DNA exists as a separate molecule within the bacterial cell, and replicates separately from the host bacterial DNA.
  • The T-4&rsquos tail fibres allow attachment to a host cell, and the T4&rsquos tail is hollow so that it can pass its nucleic acid to the cell it is infecting during attachment. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.
  • lytic cycle: The normal process of viral reproduction involving penetration of the cell membrane, nucleic acid synthesis, and lysis of the host cell.
  • virulence: the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host.

Virulence is the degree of pathogenicity within a group or species of parasites as indicated by case fatality rates and/or the ability of the organism to invade the tissues of the host. The pathogenicity of an organism is determined by its virulence factors. Virus virulence factors determine whether an infection will occur and how severe the resulting viral disease symptoms are. Viruses often require receptor proteins on host cells to which they specifically bind. Typically, these host cell proteins are endocytosed and the bound virus then enters the host cell. Virulent viruses such as HIV, which causes AIDS, have mechanisms for evading host defenses.

Some viral virulence factors confer ability to replicate during the defensive inflammation responses of the host such as during virus-induced fever. Many viruses can exist inside a host for long periods during which little damage is done. Extremely virulent strains can eventually evolve by mutation and natural selection within the virus population inside a host. The term &ldquoneurovirulent&rdquo is used for viruses such as rabies and herpes simplex which can invade the nervous system and cause disease there.

Model organisms of virulent viruses that have been extensively studied include virus T4 and other T-even bacteriophages which infect Escherichia coli and a number of related Bacteria.

The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. The lytic cycle is typically considered the main method of viral replication, since it results in the destruction of the infected cell. A key difference between the lytic and lysogenic phage cycles is that in the lytic phage, the viral DNA exists as a separate molecule within the bacterial cell, and replicates separately from the host bacterial DNA. The location of viral DNA in the lysogenic phage cycle is within the host DNA, therefore in both cases the virus/phage replicates using the host DNA machinery, but in the lytic phage cycle, the phage is a free floating separate molecule to the host DNA.

Figure: Cycles of viral reproduction: Comparison of the bacteriophage lysogenic and lytic cycles.

The lytic cycle is a six-stage cycle. In the first stage, called &ldquopenetration,&rdquo the virus injects its own nucleic acids into a host cell. Then the viral acids form a circle in the center of the cell. The cell then mistakenly copies the viral acids instead of its own nucleic acids. Then the viral DNA organize themselves as viruses inside the cell. When the number of viruses inside becomes too much for the cell to hold, the membrane splits and the viruses are free to infect other cells. Some viruses escape the host cell without bursting the cell membrane instead, they bud off from it by taking a portion of the membrane with them. Because it otherwise is characteristic of the lytic cycle in other steps, it still belongs to this category, although it is sometimes named the Productive Cycle. HIV, influenza and other viruses that infect eukaryotic organisms generally use this method.

T-4 bacteriophage is a bacteriophage that infects E. coli bacteria. Its double-stranded DNA genome is about 169 kbp long and is held in an icosahedral head, also known as a capsid. T4 is a relatively large phage, at approximately 90 nm wide and 200 nm long (most phages range from 25 to 200 nm in length). Its tail fibres allow attachment to a host cell, and the T4&rsquos tail is hollow so that it can pass its nucleic acid to the cell it is infecting during attachment. T4 is capable of undergoing only a lytic lifecycle and not the lysogenic lifecycle.

The T4 Phage initiates an E. coli infection by recognizing cell surface receptors of the host with its long tail fibers (LTF). A recognition signal is sent through the LTFs to the baseplate. This unravels the short tail fibers (STF) that bind irreversibly to the E. coli cell surface. The baseplate changes conformation and the tail sheath contracts causing GP5 at the end of the tail tube to puncture the outer membrane of the cell. The lysozyme domain of GP5 is activated and degrades the periplasmic peptidoglycan layer. The remaining part of the membrane is degraded and then DNA from the head of the phage can travel through the tail tube and enter the E. coli.

The lytic lifecycle (from entering a bacterium to its destruction) takes approximately 30 minutes (at 37 °C) and consists of:

  • Adsorption and penetration (starting immediately)
  • Arrest of host gene expression (starting immediately)
  • Enzyme synthesis (starting after 5 minutes)
  • DNA replication (starting after 10 minutes)
  • Formation of new virus particles (starting after 12 minutes)

After the life cycle is complete, the host cell bursts open and ejects the newly built viruses into the environment, destroying the host cell. T4 has a burst size of approximately 100-150 viral particles per infected host. Complementation, deletion, and recombination tests can be used to map out the rII gene locus by using T4. These bacteriophage infect a host cell with their information and then blow up the host cell, thereby propagating themselves.

Molecular weights of coliphages and coliphage DNA : I. Measurement of the molecular weight of bacteriophage T7 by high-speed equilibrium centrifugation☆

The range of usefulness of the high-speed equilibrium centrifugation method of Yphantis (1964) has been extended to measure the molecular weight of Escherichia coli phage T7. Values for ̄ gn of phages T7, T5 and T4 were obtained by pycnometry the phage concentrations were determined by measuring nitrogen and phosphorus contents, and the amino-acid and nucleotide composition. The ̄ gn values are: T7, 0.639 T5, 0.658 and T4, 0.618. From the nitrogen and phosphorus measurements, the percentage DNA in each phage type has been calculated. These values are: T7, 51.2 T5, 61.7 and T4, 54.9. The molecular weights of T7 phage and T7 DNA are 49.4 and 25.3 million daltons, respectively. In the following paper (Dubin, Benedek, Bancroft & Freifelder, 1970), values are reported of the molecular weights of T7, T5 and T4, determined by another technique. The values yielded by the two techniques for the molecular weight of T7 are in excellent agreement. Discussion of the general problem of molecular weight measurement is given in Freifelder (1970). ‡

This is publication no. 742 from the Graduate Department of Biochemistry, Brandeis University.

Present address: Department of Biological Sciences, Columbia University, New York, N.Y. 10027, U.S.A.

A prelimenary report of these findings has been made (Bancroft & Freifelder, 1969)

Types of tRNA

A tRNA can be classified based on the amino acid it carries, giving rise to 20 different tRNAs. Alternatively, they can also be grouped based on their anticodon. There are 64 possible codons arising from a combination of four nucleotides. Of these, 3 are stop codons that signal the end of translation. This gives rise to a situation where one amino acid is represented by multiple codons and the AATS, as well as the tRNAs have to accommodate this redundancy. However, very few species have exactly 61 tRNAs, which gives rise to the question of how every codon is recognized by a specific tRNA. In many species, the number far exceeds 61 and different tRNAs carrying the same anticodon could display varying efficiency in translation, adding a layer of regulation to the process of protein synthesis.

tRNAs interact with codons on the mRNA through their anticodon loop. Base pairing between the codon and anticodon ensures specificity during translation. However, the first base of the anticodon, that pairs with the ‘wobble’ or third position in a codon is often modified to allow the tRNA to hydrogen bond with three, instead of one base. Thus a single tRNA has the option of recognizing and base pairing with three codons, which code for the same amino acid. There are 20 AATS, one for each amino acid. This group of enzymes can recognize all the anticodons representing a particular amino acid and therefore act as the second arm of the machinery that handles genetic code redundancy.

Finally, these molecules can also be classified into three categories – those carrying canonical amino acids attached to the correct tRNA, those that are incorrectly attached, and those carrying modified amino acids such as selenocysteine for non-canonical elongation.

Post-Transcriptional Modification of tRNA

There are nearly 500 genes coding for tRNAs in the human genome, and 300 gene fragments associated with these RNA. These genes are transcribed by RNA polymerase III and the transcript undergoes extensive modification, especially in eukaryotes. Introns are spliced, the intron-exon boundary is acted on by endonucleases, the 5’ and 3’ ends of the RNA are processed and enzymes add the terminal CCA residues to the 3’ end of the tRNA. The CCA residues could become aminoacylated in the nucleus itself and this charged tRNA could then be exported from the nucleus.

Additionally, many bases on the tRNA are also modified, especially by methylation (addition of a methyl group) and deamidation (removal of an amide group). Particularly, the first base of the anticodon that pairs with the ‘wobble’ position on the codon is modified to allow unusual types of base pairing. Adenine can be modified to form inosine, which expands the pairing possibilities to include uracil, cytosine and adenine. Pseudouridine is another common modified base, derived from uridine residues through enzyme-mediated isomerization. It is said to play a role in the structural integrity of the tRNA molecule, being involved in stiffening the nearby sugar-phosphate backbone and also influencing base stacking of proximal regions. Lysidine is an unusual base formed when a lysine amino acid is attached to cytidine residue. Lysidine pairs specifically with adenosine, a property that is used by the isoleucine tRNA to ensure translation specificity.

AATS attach the appropriate amino acid to tRNA molecules based on their anticodon. These enzymes contain binding sites for the amino acid, tRNA as well as ATP and hydrolyze ATP to AMP and attach the amino acid to the ribose sugar of the last nucleotide on tRNA. The tRNA is now considered ‘charged’ and can participate in the protein synthesizing reactions on the ribosome. This reaction often occurs in the cytoplasm, though it has also been observed in the nucleus.

The enzyme binds to many regions of the tRNA to ensure high specificity in the reaction and even proofreads its own reaction since many amino acids have similar structures.

Mature tRNA then binds specific export factors that export it from the nucleus, using the RanGTP system. The acceptor arm and T-arm play an important role in this process, and there is extensive interaction between the export factors and the RNA molecule, allowing only fully processed, complete tRNAs to move to the cytoplasm.

Viral Genomes | Chromosome

Viruses are a special class of infectious agents that are so small that they can be viewed only under electron microscope. A complete “viral particle” or “virion consists of a block of genetic material (DNA or RNA) surrounded by a protein coat and, sometimes by an additional membranous envelope.

The viruses contain neither cytoplasm nor exhibit any growth or metabolic activity. But when their genetic material enters into a suitable host cell, virus-specific protein synthesis replication of the viral chromosome occurs these processes utilize both cellular (of host) and viral enzymes.

On the basis of the host organisms, viruses are divided into three main groups:

Morphological Features of Viruses:

The viral chromosome is enclosed within a protein shell called capsid. The viral chromosome and its protein coat together are called nucleocapsid. Viruses vary considerably in their morphological features (Table 5.4).

1. Icosahedral virions:

Their capsid is icosahedral, i.e., the virion is a regular polyhedron with 20 triangular faces and 12 corners. Examples are, adenoviruses and bacteriophage φX174.

2. Helical virions:

The nucleic acid of such virions is enclosed in a cylindrical, rod shape capsid that forms a helical structure, e.g., TMV, bacteriophage M13.

3. In some cases, the nucleocapsid is icosahedral while in others, it is helical in some components. Such viruses are enveloped.

These viruses do not have a clearly identifiable capsid. The viral nucleic acid is present in the centre of the shell which is made up of protein molecules. Some of the shells are complex while others are simple. In Herpes, an animal virus that contains DNA as genetic material, the capsid has a diameter of 1000A it is further surrounded by an envelope making its diameter 1500A. (Fig. 5.19).

The capsid is mode up of protein subunits (capsomers) which form an icosahedron.

Bacteriophages have relatively complex structures: they contain a head, a tail, a base plate and several tail fibres (Fig. 5.20). The head is hexagonal (lateral side) and contains the viral DNA. The tail has a core tube surrounded by a sheath. At the tail end, there is a basal plate with 6 spikes from which 6 tail fibres emerge.

At the time of infection, the tail fibres bind to specific receptor sites on the host cell. The base plate is drawn to the cell surface and contraction of tube sheath occurs along with the removal of the base plate plug. The core of the tail penetrates the cell wall which is weakened by some hydrolytic enzymes present in the phage and the viral tail. DNA enters into the host cell through the core tube of the tail.

In the case of tobacco mosaic virus (TMV multiplying in tobacco plant cells) and some small bacterial viruses (e.g., F2, R17, QB), the protein coat contains a single type of protein. These protein molecules are arranged in either a helical symmetry or a cubical symmetry.

The shell of TMV contains about 2150 protein molecules which are identical, each molecule having the molecular weight of-17,000. These molecules are helically arranged around the RNA genome which contains 6,000 nucleotides.

The viruses which lyse or disrupt the host cell following infection are called lytic viruses. During infection, the nucleic acid is injected into the host cell. The enzymes required for viral DNA replication are then synthesized so that replication of DNA occurs to produce numerous copies of the viral chromosome.

The protein components of the capsid are synthesized in the later stages leading to the formation of heads and tails the viral DNA is then packed into the heads. In the end, the cell wall ruptures and the progeny phage particles are released (Fig. 5.21).

Lysogenic Viruses (Temperate Phages):

Lysogeny involves a symbiotic relationship between a temperate phage and its bacterial host. The viral chromosome becomes inserted into the bacterial chromosome, where it remains and replicates along with the latter. The viral DNA integrated into the bacterial genome is called a provirus or prophage (Fig. 5.22). The bacterium containing a prophage is immune to the infection by the same virus.

Viral Chromosomes:

Viruses contain either DNA or RNA as their genetic material. These nucleic acids may be either single or double-stranded (Table 5.5). Small viruses may contain 3 kb (kb =,kilo-bases = 1000 bases), while large viruses could have about 300 kb. in their genome. Thus the number of genes in viral genome may vary from only 3 to hundreds. The retroviruses arc diploid (have two copies of the genome per capsid), while the others are haploid.

Several viruses possess double-stranded DNA as their genetic material. The base composition of different viruses is modified leading to change in the physical properties of DNA such melting temperature, buoyant density in caesium chloride (CsCl) etc.

In some of the viruses, such as. T-even coliphages, cytosine (C) is modified into 5-hydroxymethyl- cytosine (HMC). In certain cases, thymine is converted into 5-hydroxy-methyl uracil or 5-di-hydroxymethyluracil, e.g., in B. subtilisbacteriophges. Certain physical properties of DNA, such as, buoyant density in CsCl or melting temperature are changed due to these substitutions.

Some of the viruses contain linear DNA, while others contain circular (cyclic) DNA (Table 5.5). In the case of phage lambda (λ), DNA can exist in both linear and cyclic forms. When isolated from a viral particle, the λ DNA is linear, but when it enters into the host cell, becomes circular. However, it enters into the host cell in its linear form.

The A. chromosome is a double- stranded DNA molecule containing 47,000 nucleotides it is 17 pm in length. There is single- stranded projection of 12 nucleotides at each 5′-end these projections are complementary to each other and thus they are called cohesive ends.

These cohesive ends are responsible for the circularization of the chromosome. Circularization of the chromosome protects it from degradation by the host exonucleases. Further, the linear DNA cannot replicate vegetatively the circularity therefore, provides an advantage in replication as well.

Single-stranded DNA occurs in very small bacteriophages (Table 5.4). The single-stranded DNA found in the virion is called the positive (+) strand as a rule only the plus (+) strand is found in the phage particles. However, in adeno-associated viruses, two complementary strands exist in different virions. The single-stranded DNA contains inverted repeating sequences that form hair pins. The hairpin structures have important role in circularization of the linear strands and in replication.

Double-stranded RNAs are found in several icosahedral viruses of animals and plants. The genomes of such viruses are segmented (Table 5.5). The different segments may be connected short stretches of base pairs. Transcription of each segment occurs separately and the enzyme involved is “Double-stranded RNA transcriptase”. Each mRNA, on translation produces a separate polypeptide chain.

Single-stranded RNA is the genetic material in a number of viruses (Table 5.5). Some viruses contain a single RNA molecule in their genome, while some other viruses contain several segments, e.g., influenza virus has 8 segments. The viruses contain either positive (+) or negative (-) strands of RNA in their capsids.

The viral RNA strand that functions as mRNA in the host cell is called the plus (+) strand or positive strand. The RNA genomes of animal viruses have a cap at their 5′-end and a poly (A) sequence at the 3′-end. However, in Picornavirus RNA, there is a special sequence at the 5′-end to which a small protein is covalently attached.

The RNA genomes of plant viruses possess a cap at the 5′-end but they do not contain the poly (A) at their 3′-ends their 3′-end is similar to tRNA. Each retrovirus particle contains two copies of the (+) RNA strand representing its genome these copies are held together near the 5′-end.

These RNAs do not contain a cap but terminate into a nucleoside triphosphate at their 5′-ends. These strands do not function as mRNA directly. Instead, they are transcribed by the enzyme “single-stranded RNA transcriptase” present in the virion, to produce the mRNA.

Packaging of Nucleic Acids in the Viruses:

Viral genome (DNA/RNA) is tightly packed into the protein shell (capsid). The density of the nucleic acid in the protein shell is higher than 500 mg/ml, which is much greater than the density of DNA in other organisms. For example, density of DNA in bacterium is about 10 mg/ml, while in the eukaryotic nucleus, it is about 100 mg/ml. This shows that the nucleic acid is very tightly packaged in the viral particles.

The genetic material of TMV is single-stranded RNA containing 6400 nucleotides, making up a length of 2 pm. This RNA is packaged into the rod-shaped compartment of 0.3 x 0.008 pm. Adenoviruses contain 11 pm long double-stranded DNA consisting of 35,000 bp: this is packaged into an icosahedron type capsid of 0.07 pm diameter.

Phage T4 has a very long double-stranded DNA molecule (55 pm) having 170,000 bp. The capsid containing this rather long DNA is an icosahedron with the dimensions of 1.0 x 0.065 pm. Unlike eukaryotic nucleus and bacterial nucleoid, the volume of the capsid is fully packaged with the nucleic acid.

Packaging of nucleic acid to form a nucleocapsid occurs in two general ways. In one mechanism, the protein molecules assemble around the nucleic acid, e.g., in TMV. In the other mechanism, the protein coat is formed first and then the nucleic acid is inserted in it. In TMV, a duplex hairpin structure occurs in the RNA.

The assembly of protein monomers begins at this nucleation centre and proceeds in both the directions, reaching the ends. A total of 17 protein units form a circular layer and two such layers together form a unit of capsid. This structure interacts with the RNA which is coiled to form a helix inside the shell.

In bacteriophage T4 and λ etc., the protein shell is formed first. The nucleic acid is inserted into the coat from one end and then the tail is joined to the head. In case of circular DNA, it must be first converted into a linear molecule for packaging.

The lambda (λ) genome is circular and contains two “cos” sites, cosL and cosR. The free end in λ DNA is produced by enzymatic cleavage at the cosL site. Insertion of DNA occurs from this end and continues till the cosR site enters the capsid a cleavage then occurs at the cosR site to produce the other end of the λ genome.

Some of the viruses, e.g., phage T4 and λ. have terminal redundancy in their genomes. In these viruses, multiple genomes join end-to-end to produce “concatemeric structure.” In case of T4, insertion of the viral chromosome starts at a “random” point and continues until the required amount of DNA has been inserted into the head. The DNA inserted into the head has a terminal redundancy.

One likely origin of the “concatermeric” DNA is recombination. Recombination between two chromosomes combines two genomes end-to-end. Then recombination with a third genome produces a concatemer through successive recombination’s (Fig. 5.23).

Another mechanism suggested for concatemer formation is the rolling circle replication. Specific endonuclease cuts the concatemer at the points that produce the genome of the “required length.” The genomic DNA has homologous ends due to the terminal redundancy. Therefore, some chromosomes may be heterozygous for the terminal genes.

Mechanisms of Lysogenic and Lytic Pathways:

Bacteriophage λ is a temperate phage that maintains a lysogenic relationship with its bacterial host. However, it can undergo lytic cycle also. Infection, as a rule, occurs in the linear form, but the chromosome converts into a circular one once it enters the host cell. A generalized map of the X chromosome showing different functions is presented in Fig. 5.24.

Genes related to similar functions are clustered. On the linear chromosome, genes for head formation are located on left end, while those for lysis are located at the right end. The regulatory region lies between the region for recombination and the region for replication. The genes present in the regulatory region are responsible for determining whether the X will enter into a lysogenic relationship with its host or it will follow the lytic pathway.

Regulatory genes are clustered and flanked by genes for recombination on their left side and those for replication on the right side (Fig. 5.25). Genes N (anti-terminator) and era (anti-repressor) are located within the regulatory region. These genes are called “immediate early genes” they are transcribed by the host RNA polymerase.

In the presence of anti-termination factor (p N ), transcription of both the genes (N and era) continues. These two genes are transcribed from different DNA strands in the opposite direction, the gene N being transcribed towards the left, while era is transcribed towards the right.

The transcription extends to other region of the genome for different functions (Fig. 5.25). In the absence of cl repressor protein, the host RNA polymerase binds to PL/OL sites so that the transcription of the “late genes” is initiated as a result, phage particles are produced and the cell is lysed.

The regulatory region contains the cl gene which is responsible for the lysogenic pathway. A mutation in this region causes the phage to undergo lytic cycle.

The cl gene is transcribed to produce mRNA the enzyme involved in transcription is RNA polymerase that binds to the promoter for repressor maintenance (PRM). The transcription occurs from right to left. This cl mRNA is translated to produce the repressor monomer (Fig. 5.25).

Repressor dimers are formed that bind to the PL/OR and PL/OL sites, thus preventing the RNA polymerase from binding to these promoters. This leads to the inhibition of transcription of N and cro genes. Later, the X chromosome is integrated into the bacterial chromosome its delayed early genes are not expressed and the phage remains as a “provirus”. Delayed early genes are the genes for recombination, replication and Q (anti-terminator). Late genes are tail, head and lysis genes.

When the cl repressor is bound to the 0L and 0R sites, RNA polymerase initiates transcription of the cl gene, and synthesis of repressor protein is continued. But in absence of the repressor, RNA polymerase binds to PL/OL and Pr/Or sites and transcription of N and cro genes begins.

Thus the presence of cl repressor itself is necessary for its synthesis. Continuous production of cl repressor is necessary for lysogeny to be maintained. During this period, the OL and OR sites are always bound by repressor.

When the lysogenized cell is infected by another phage X, the cl repressor protein produced by the “prophage” immediately binds to the OL and 0R sites of the infecting X genome. The function of the infecting X genes is thus inhibited and the cell remains immune to X infection.


The present study aimed to determine the differences in the behaviors of four F-specific RNA (F-RNA) coliphage genogroups (GI–GIV) during wastewater treatment. Raw sewage, aeration tank effluent, secondary-treated sewage, and return activated sludge were collected from a wastewater treatment plant in Japan at monthly intervals between March and December 2011 (n = 10 each). F-specific coliphages were detected by plaque assay in all tested samples, with a concentration ranging from − 0.10 to 3.66 log10 plaque-forming units/ml. Subsequently, eight plaques were isolated from each sample, followed by genogroup-specific reverse-transcription quantitative PCR (qPCR) for F-RNA coliphages and qPCR for F-specific DNA (F-DNA) coliphages. GI F-RNA coliphages were the most abundant in the secondary-treated sewage samples (73% of the plaque isolates), while GII F-RNA coliphages were the most abundant in the other three sample types (41–81%, depending on sample type). Based on the results of the quantification and genotyping, the annual mean concentrations of each F-specific coliphage type were calculated, and their reduction ratios during wastewater treatment were compared with those of indicator bacteria (total coliforms and Escherichia coli) and enteric viruses (human adenoviruses and GI and GII noroviruses). The mean reduction ratio of GI F-RNA coliphages was the lowest (0.93 log10), followed by those of the indicator bacteria and enteric viruses (1.59–2.43 log10), GII–GIV F-RNA coliphages (> 2.60–3.21 log10), and F-DNA coliphages (> 3.41 log10). These results suggest that GI F-RNA coliphages may be used as an appropriate indicator of virus reduction during wastewater treatment.

Correlated (or Paired) T-Test

The correlated t-test is performed when the samples typically consist of matched pairs of similar units, or when there are cases of repeated measures. For example, there may be instances of the same patients being tested repeatedly—before and after receiving a particular treatment. In such cases, each patient is being used as a control sample against themselves.

This method also applies to cases where the samples are related in some manner or have matching characteristics, like a comparative analysis involving children, parents or siblings. Correlated or paired t-tests are of a dependent type, as these involve cases where the two sets of samples are related.

The formula for computing the t-value and degrees of freedom for a paired t-test is:

The remaining two types belong to the independent t-tests. The samples of these types are selected independent of each other—that is, the data sets in the two groups don’t refer to the same values. They include cases like a group of 100 patients being split into two sets of 50 patients each. One of the groups becomes the control group and is given a placebo, while the other group receives the prescribed treatment. This constitutes two independent sample groups which are unpaired with each other.


Study sites and sample collection.

Human and animal wastewater, freshly voided animal fecal samples, and surface waters potentially impacted by waste discharges or runoff from a variety of well-defined land uses were collected and analyzed by The University of North Carolina (UNC) Department of Environmental Sciences and Engineering and the University of Massachusetts (UMass) Department of Civil and Environmental Engineering. Samples were collected from surface water sites monthly for 40 months. A subset of surface water samples was also collected during or just following precipitation events (storm samples). At UNC, storm samples were collected with ISCO (Lincoln, Nebr.) automatic samplers that were triggered when the stream height increased by 0.5 in. Analyzed storm samples represented a composite of the storm hydrograph. Storm samples were collected manually at UMass within 24 h of a precipitation event that exceeded 0.1 in.

Freshly voided feces (50 g) and liquid wastewater samples (500 ml) were collected aseptically from a variety of feral and domestic animals, from cattle and swine waste lagoons, and from human wastewater treatment plants (WWTP). In addition, surface waters (2-liter samples) were collected from sites identified as being potentially impacted by urban or rural human land use (municipal sewage effluents or septic systems, respectively) or agricultural land use (swine or cattle farms). For each surface water study site, an upstream or background station was identified and sampled on the same day. All samples were collected and transported to the laboratory in sterile, wide-mouth, high-density polyethylene bottles on ice or commercial freezer packs and analyzed within 24 h (WWTP, waste lagoon, and surface water samples) or 72 h (solid wastes) of sample collection. Data recorded at the time of analysis included the sampling site, animal species, or waste source associated with the sample or sampling site, the date of collection, and whether the sample was collected during a storm event.

F+ coliphage isolation and serotyping.

F+ coliphages were enumerated by direct plating of serial dilutions (wastes and wastewater) or cellulose membrane filter adsorption-elution concentration (surface water), followed by double or single agar layer plaque assay methods (U.S. Environmental Protection Agency method 1602) (29, 30). When available, up to 10 coliphage isolates were removed from the sample agar, suspended in phosphate-buffered saline (PBS) containing 20% glycerol, and stored at �ଌ until further analysis. F+ RNA and DNA coliphages were distinguished by spotting (5 μl) and incubation of serial dilutions (10 𢄢 , 10 𢄤 , and 10 𢄦 ) of the isolated F+ coliphage suspended in PBS on nutrient agar-host (E. coli Famp) plates (control) or nutrient agar-host plates containing RNase (experimental) for 12 to 16 h. Phage growth on both the control and experimental plates at all dilutions was indicative of F+ DNA phage. A type strain group I coliphage, MS2 (previously molecularly characterized), was used as a positive F+ RNA control, and PBS was used as a negative control. F+ RNA phage isolates were serotyped to group phages into the following categories: group I (MS2), group II (GA), group III (Qβ), and group IV (SP). Briefly, serial dilutions of the field F+ RNA isolate were plated in 5-μl spots on nutrient agar-E. coli Famp host plates containing neutralizing antisera to MS2, GA, Qβ, or SP coliphages. Failure to propagate at all dilutions in the presence of an antiserum was recorded as a positive serogroup identification.

Statistical analysis.

The results of all isolate evaluations were entered into a database and examined for bivariate associations with recorded sample data. When stream data were entered into the database, zero was entered when F+ coliphages were below the detection limit. When the density of coliphages detected exceeded the countable range, no density entry was made in the database however, a second variable was assigned that recorded whether phages were detected or not. The distribution of the stream coliphage density data (PFU per liter) was evaluated for log normalcy with a Kolmogorov Smirnov test prior to statistical testing. This distribution was used to estimate the density of coliphages among samples that exceeded the countable range. Paired t tests were used to compare the log10 geometric means of the density data grouped by land use impact. A chi-square or Fisher exact test was used to evaluate potentially significant associations between frequencies of coliphage detection and proportions of coliphage serogroups among land use categories. All statistical tests were evaluated at the 95% confidence level.

Presenting the results of a t-test

When reporting your t-test results, the most important values to include are the t-value, the p-value, and the degrees of freedom for the test. These will communicate to your audience whether the difference between the two groups is statistically significant (a.k.a. that it is unlikely to have happened by chance).

You can also include the summary statistics for the groups being compared, namely the mean and standard deviation. In R, the code for calculating the mean and the standard deviation from the data looks like this: %>%
group_by(Species) %>%
summarize(mean_length = mean(Petal.Length),
sd_length = sd(Petal.Length))

In our example, you would report the results like this:

Luria-Delbrück & the Fluctuation Test

A phenomenon that was observed from the very early days of d'Herelle's work was the occurrence, on bacterial plates that were infected with high amounts of phages so as to completely lyse the bacteria, of bacterial colonies that were resistant to phage infection. Ultimately, the question became “Are these bacterial mutants that exist prior to phage infection, or is the resistance somehow caused by the infection process itself, in a few of the bacteria?”

Salvador Luria (1984) conceived of an experiment that would distinguish these possibilities. Luria reasoned that if he divided a bacterial culture into multiple aliquots and grew them individually, then, if mutation happened to bacteria without the presence of phages, the process would occur randomly some of these cultures would have no mutations, but in others mutation might occur soon after growth began, and those mutants would reproduce throughout the incubation of the culture, producing a clone of phage-resistant bacteria. If he then plated these separate cultures along with high numbers of phages, cultures that had no or late-occurring mutations would show, on average, only a few resistant colonies. However, any cultures in which mutation occurred early in the incubation period would show a large number of phage-resistant colonies because the early-forming mutants had time to produce many descendants during the incubation.

If, on the other hand, resistance to phage lysis occurred only by interaction with the phage, those events would be rare and independent, and their distribution among the bacterial plates would follow the Poisson distribution – with no plates showing large numbers of phage-resistant colonies. Luria performed the experiment multiple times, and the distribution showed occasional plates with very large numbers of phage-resistant colonies, the expected result for the hypothesis that bacteria mutate to phage resistance independent of the presence of phages. Delbrück, with whom Luria had been collaborating, formalized the mathematical treatment and published the work, now known as the “fluctuation test” (Luria & Delbrück, 1943). This paper was a watershed in the development of molecular biology because it showed the existence of independent mutation in bacteria (Luria, 2007).

Transgender Ideology Is Riddled With Contradictions. Here Are the Big Ones.


Former Senior Research Fellow

Now, activists claim that gender identity is destiny, while biological sex is the social construct. itakdalee/Getty Images

People say that we live in a postmodern age that has rejected metaphysics. That’s not quite true.

We live in a postmodern age that promotes an alternative metaphysics. As I explain in “When Harry Became Sally,” at the heart of the transgender moment are radical ideas about the human person—in particular, that people are what they claim to be, regardless of contrary evidence. A transgender boy is a boy, not merely a girl who identifies as a boy.

It’s understandable why activists make these claims. An argument about transgender identities will be much more persuasive if it concerns who someone is, not merely how someone identifies. And so the rhetoric of the transgender moment drips with ontological assertions: People are the gender they prefer to be. That’s the claim.

Transgender activists don’t admit that this is a metaphysical claim. They don’t want to have the debate on the level of philosophy, so they dress it up as a scientific and medical claim. And they’ve co-opted many professional associations for their cause.

Thus the American Psychological Association, in a pamphlet titled “Answers to Your Questions about Transgender People, Gender Identity, and Gender Expression,” tells us, “Transgender is an umbrella term for persons whose gender identity, gender expression, or behavior does not conform to that typically associated with the sex to which they were assigned at birth.”

Notice the politicized language: A person’s sex is “assigned at birth.” Back in 2005, even the Human Rights Campaign referred instead to “birth sex” and “physical sex.”

The phrase “sex assigned at birth” is now favored because it makes room for “gender identity” as the real basis of a person’s sex.

In an expert declaration to a federal district court in North Carolina concerning H.B. 2, Dr. Deanna Adkins stated, “From a medical perspective, the appropriate determinant of sex is gender identity.” Adkins is a professor at Duke University School of Medicine and the director of the Duke Center for Child and Adolescent Gender Care (which opened in 2015).

Adkins argues that gender identity is not only the preferred basis for determining sex, but “the only medically supported determinant of sex.” Every other method is bad science, she claims: “It is counter to medical science to use chromosomes, hormones, internal reproductive organs, external genitalia, or secondary sex characteristics to override gender identity for purposes of classifying someone as male or female.”

This is a remarkable claim, not least because the argument recently was that gender is only a social construct, while sex is a biological reality. Now, activists claim that gender identity is destiny, while biological sex is the social construct.

Adkins doesn’t say if she would apply this rule to all mammalian species. But why should sex be determined differently in humans than in other mammals? And if medical science holds that gender identity determines sex in humans, what does this mean for the use of medicinal agents that have different effects on males and females? Does the proper dosage of medicine depend on the patient’s sex or gender identity?

But what exactly is this “gender identity” that is supposed to be the true medical determinant of sex? Adkins defines it as “a person’s inner sense of belonging to a particular gender, such as male or female.”

Note that little phrase “such as,” implying that the options are not necessarily limited to male or female. Other activists are more forthcoming in admitting that gender identity need not be restricted to the binary choice of male or female, but can include both or neither. The American Psychological Association, for example, defines “gender identity” as “a person’s internal sense of being male, female, or something else.”

Adkins asserts that being transgender is not a mental disorder, but simply “a normal developmental variation.” And she claims, further, that medical and mental health professionals who specialize in the treatment of gender dysphoria are in agreement with this view.

Transgender Catechism

These notions about sex and gender are now being taught to young children. Activists have created child-friendly graphics for this purpose, such as the “Genderbread Person.” The Genderbread Person teaches that when it comes to sexuality and gender, people have five different characteristics, each of them falling along a spectrum.

There’s “gender identity,” which is “how you, in your head, define your gender, based on how much you align (or don’t align) with what you understand to be the options for gender.” The graphic lists “4 (of infinite)” possibilities for gender identity: “woman-ness,” “man-ness,” “two-spirit,” or “genderqueer.”

The second characteristic is “gender expression,” which is “the way you present gender, through your actions, dress, and demeanor.” In addition to “feminine” or “masculine,” the options are “butch,” “femme,” “androgynous,” or “gender neutral.”

Third is “biological sex,” defined as “the physical sex characteristics you’re born with and develop, including genitalia, body shape, voice pitch, body hair, hormones, chromosomes, etc.”

The final two characteristics concern sexual orientation: “sexually attracted to” and “romantically attracted to.” The options include “Women/Females/Femininity” and “Men/Males/Masculinity.” Which seems rather binary.

The Genderbread Person tries to localize these five characteristics on the body: gender identity in the brain, sexual and romantic attraction in the heart, biological sex in the pelvis, and gender expression everywhere.

The Genderbread Person espouses the latest iteration of transgender ideology. (Photo: Sam Killerman/It’s Prounounced Metrosexual)

The Genderbread Person presented here is version 3.3, incorporating adjustments made in response to criticism of earlier versions. But even this one violates current dogma. Some activists have complained that the Genderbread Person looks overly male.

A more serious fault in the eyes of many activists is the use of the term “biological sex.” Time magazine drew criticism for the same transgression in 2014 after publishing a profile of Laverne Cox, the “first out trans person” to be featured on the cover.

At least the folks at Time got credit for trying to be “good allies, explaining what many see as a complicated issue,” wrote Mey Rude in an article titled “It’s Time for People to Stop Using the Social Construct of ‘Biological Sex’ to Defend Their Transmisogyny.” (It’s hard to keep up with the transgender moment.)

But Time was judged guilty of using “a simplistic and outdated understanding of biology to perpetuate some very dangerous ideas about trans women,” and failing to acknowledge that biological sex “isn’t something we’re actually born with, it’s something that doctors or our parents assign us at birth.”

Today, transgender “allies” in good standing don’t use the Genderbread Person in their classrooms, but opt for the “Gender Unicorn,” which was created by Trans Student Educational Resources. It has a body shape that doesn’t appear either male or female, and instead of a “biological sex” it has a “sex assigned at birth.”

Those are the significant changes to the Genderbread Person, and they were made so that the new graphic would “more accurately portray the distinction between gender, sex assigned at birth, and sexuality.”

According to Trans Student Education Resources, “Biological sex is an ambiguous word that has no scale and no meaning besides that it is related to some sex characteristics. It is also harmful to trans people. Instead, we prefer ‘sex assigned at birth’ which provides a more accurate description of what biological sex may be trying to communicate.”

The Gender Unicorn is the graphic that children are likely to encounter in school. These are the dogmas they are likely to be catechized to profess.

The Gender Unicorn is used to avoid using a male or female body as default. (Photo: Landyn Pan and Anna Moore/Trans Student Educational Resources)

While activists claim that the possibilities for gender identity are rather expansive—man, woman, both, neither—they also insist that gender identity is innate, or established at a very young age, and thereafter immutable.

Dr. George Brown, a professor of psychiatry and a three-time board member of the World Professional Association for Transgender Health, stated in his declaration to the federal court in North Carolina that gender identity “is usually established early in life, by the age of 2 to 3 years old.”

Addressing the same court, Adkins asserted that “evidence strongly suggests that gender identity is innate or fixed at a young age and that gender identity has a strong biological basis.” (At no point in her expert declaration did she cite any sources for any of her claims.)

Transgender Contradictions

If the claims presented in this essay strike you as confusing, you’re not alone. The thinking of transgender activists is inherently confused and filled with internal contradictions. Activists never acknowledge those contradictions. Instead, they opportunistically rely on whichever claim is useful at any given moment.

Here I’m talking about transgender activists. Most people who suffer from gender dysphoria are not activists, and many of them reject the activists’ claims. Many of them may be regarded as victims of the activists, as I show in my book.

Many of those who feel distress over their bodily sex know that they aren’t really the opposite sex, and do not wish to “transition.” They wish to receive help in coming to identify with and accept their bodily self. They don’t think their feelings of gender dysphoria define reality.

But transgender activists do. Regardless of whether they identify as “cisgender” or “transgender,” the activists promote a highly subjective and incoherent worldview.

On the one hand, they claim that the real self is something other than the physical body, in a new form of Gnostic dualism, yet at the same time they embrace a materialist philosophy in which only the material world exists. They say that gender is purely a social construct, while asserting that a person can be “trapped” in the wrong gender.

They say there are no meaningful differences between man and woman, yet they rely on rigid sex stereotypes to argue that “gender identity” is real, while human embodiment is not. They claim that truth is whatever a person says it is, yet they believe there’s a real self to be discovered inside that person.

They promote a radical expressive individualism in which people are free to do whatever they want and define the truth however they wish, yet they try ruthlessly to enforce acceptance of transgender ideology.

It’s hard to see how these contradictory positions can be combined. If you pull too hard on any one thread of transgender ideology, the whole tapestry comes unraveled. But here are some questions we can pose:

If gender is a social construct, how can gender identity be innate and immutable? How can one’s identity with respect to a social construct be determined by biology in the womb? How can one’s identity be unchangeable (immutable) with respect to an ever-changing social construct? And if gender identity is innate, how can it be “fluid”?

The challenge for activists is to offer a plausible definition of gender and gender identity that is independent of bodily sex.

Is there a gender binary or not? Somehow, it both does and does not exist, according to transgender activists. If the categories of “man” and “woman” are objective enough that people can identify as, and be, men and women, how can gender also be a spectrum, where people can identify as, and be, both, or neither, or somewhere in between?

What does it even mean to have an internal sense of gender? What does gender feel like? What meaning can we give to the concept of sex or gender—and thus what internal “sense” can we have of gender—apart from having a body of a particular sex?

Apart from having a male body, what does it “feel like” to be a man? Apart from having a female body, what does it “feel like” to be a woman? What does it feel like to be both a man and a woman, or to be neither?

The challenge for the transgender activist is to explain what these feelings are like, and how someone could know if he or she “feels like” the opposite sex, or neither, or both.

Even if trans activists could answer these questions about feelings, that still wouldn’t address the matter of reality. Why should feeling like a man—whatever that means—make someone a man? Why do our feelings determine reality on the question of sex, but on little else? Our feelings don’t determine our age or our height. And few people buy into Rachel Dolezal’s claim to identify as a black woman, since she is clearly not.

If those who identify as transgender are the sex with which they identify, why doesn’t that apply to other attributes or categories of being? What about people who identify as animals, or able-bodied people who identify as disabled? Do all of these self-professed identities determine reality? If not, why not?

And should these people receive medical treatment to transform their bodies to accord with their minds? Why accept transgender “reality,” but not trans-racial, trans-species, and trans-abled reality?

The challenge for activists is to explain why a person’s “real” sex is determined by an inner “gender identity,” but age and height and race and species are not determined by an inner sense of identity.

Of course, a transgender activist could reply that an “identity” is, by definition, just an inner sense of self. But if that’s the case, gender identity is merely a disclosure of how one feels. Saying that someone is transgender, then, says only that the person has feelings that he or she is the opposite sex.

Gender identity, so understood, has no bearing at all on the meaning of “sex” or anything else. But transgender activists claim that a person’s self-professed “gender identity” is that person’s “sex.”

The challenge for activists is to explain why the mere feeling of being male or female (or both or neither) makes someone male or female (or both or neither).

Gender identity can sound a lot like religious identity, which is determined by beliefs. But those beliefs don’t determine reality. Someone who identifies as a Christian believes that Jesus is the Christ. Someone who identifies as a Muslim believes that Muhammad is the final prophet. But Jesus either is or is not the Christ, and Muhammad either is or is not the final prophet, regardless of what anyone happens to believe.

So, too, a person either is or is not a man, regardless of what anyone—including that person—happens to believe. The challenge for transgender activists is to present an argument for why transgender beliefs determine reality.

Determining reality is the heart of the matter, and here too we find contradictions.

On the one hand, transgender activists want the authority of science as they make metaphysical claims, saying that science reveals gender identity to be innate and unchanging. On the other hand, they deny that biology is destiny, insisting that people are free to be who they want to be.

Which is it? Is our gender identity biologically determined and immutable, or self-created and changeable? If the former, how do we account for people whose gender identity changes over time? Do these people have the wrong sense of gender at some time or other?

And if gender identity is self-created, why must other people accept it as reality? If we should be free to choose our own gender reality, why can some people impose their idea of reality on others just because they identify as transgender?

The challenge for the transgender activist is to articulate some conception of truth as the basis for how we understand the common good and how society should be ordered.

As I document in depth in “When Harry Became Sally,” the claims of transgender activists are confusing because they are philosophically incoherent. Activists rely on contradictory claims as needed to advance their position, but their ideology keeps evolving, so that even allies and LGBT organizations can get left behind as “progress” marches on.

At the core of the ideology is the radical claim that feelings determine reality. From this idea come extreme demands for society to play along with subjective reality claims. Trans ideologues ignore contrary evidence and competing interests, they disparage alternative practices, and they aim to muffle skeptical voices and shut down any disagreement.

The movement has to keep patching and shoring up its beliefs, policing the faithful, coercing the heretics, and punishing apostates, because as soon as its furious efforts flag for a moment or someone successfully stands up to it, the whole charade is exposed. That’s what happens when your dogmas are so contrary to obvious, basic, everyday truths.

A transgender future is not the “right side of history,” yet activists have convinced the most powerful sectors of our society to acquiesce to their demands. While the claims they make are manifestly false, it will take real work to prevent the spread of these harmful ideas.