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In the GAL1 gene promoter system used in the yeast cross-species complementation experiment, the inducing sugar galactose promotes gene expression from the engineered construct, whereas glucose provides a noninducing comparison. The comparison concerns an imposed regulatory system, not the donor species’ native regulation. ^[ref]

Gene expression uses a gene's information to produce functional RNA or a protein. Transcription makes RNA from a DNA template; for a protein-coding gene, biological translation then uses messenger RNA to specify an amino acid chain. Many genes instead produce functional RNA, including transfer RNA and ribosomal RNA, without a translated protein product. See Alberts et al., Molecular Biology of the Cell, 4th edition (2002): from DNA to RNA.

Follow the chosen coding strand $5^{\prime }\text{-}\mathrm{ATGGCTTAA}\text{-}{3}^{\prime }$: transcription of its complementary template gives $5^{\prime }\text{-}\mathrm{AUGGCUUAA}\text{-}{3}^{\prime }$ in messenger RNA. In the stated reading frame, its three codons are AUG, GCU and UAA: methionine, alanine and a stop codon. Thus nine nucleotides specify two amino acid residues followed by termination, not three residues. This is an illustrative coding segment embedded in suitable expression and initiation context, not a claim that an isolated nine-nucleotide RNA is sufficient to initiate biological translation.

The distinction between copying and use explains how biological cells can preserve the same DNA yet produce different products. DNA replication duplicates the record; gene expression selects and uses parts of it. Transcription does not use all DNA at once, and gene expression can be controlled at several steps, including transcription, RNA processing and biological translation. See Clark, Douglas and Choi, Biology 2e: regulation of gene expression. The arrows describe template-directed synthesis: the DNA does not physically turn into a protein, and the messenger RNA does not supply the amino acids from which that protein is assembled.

Insulin is a protein hormone. Its mature form has two polypeptide chains; identifying their sequences and protein disulfide bonds provided the motivating example in Sanger's insulin sequencing experiment. See Cooper: insulin as a two-chain hormone, figure 2.16.

A meal illustrates the insulin side of blood glucose homeostasis: rising glucose stimulates beta cells in the pancreatic islets, and released insulin favors uptake and storage of fuel. Alpha cells produce glucagon, while delta cells produce somatostatin, which inhibits release of both insulin and glucagon. The pancreas therefore contains interacting sensor and secretory biological cells, rather than one undifferentiated source of hormones. ^[ref]

Insulin and glucagon are peptide hormones, built from amino acid residues joined by peptide bonds; glucose is a small carbohydrate molecule. A hormone changes signal transduction, not the identity of the fuel it regulates. Human mature insulin has 51 amino acid residues in two polypeptide chains. Beta cells process proinsulin to insulin and C-peptide, storing the products in secretory granules for exocytosis. Increased glucose metabolism raises the ATP-to-ADP ratio, tending to close ATP-sensitive potassium channels; depolarization and entry of calcium ions then trigger exocytosis. This is an important triggering pathway, with additional amplification and modulatory inputs.^[ref]

Binding to the insulin receptor, a receptor tyrosine kinase, starts phosphorylation of the receptor and insulin receptor substrates. Recruitment of phosphoinositide 3-kinase and activation of Akt transmit a major metabolic branch. In skeletal muscle and adipose tissue, that branch helps move GLUT4 to the cell membrane, increasing glucose uptake by facilitated diffusion. GLUT4 is a membrane transport protein, not an insulin receptor. Insulin also favors glycogenesis in skeletal muscle and the liver, and inhibits lipolysis in adipose tissue. In the liver, suppression of glucose output involves enzyme regulation, gene expression, and indirect changes in substrate supply. Its relative direct and indirect contributions are more complex than a single Akt arrow. ^[ref]

Insulin is not a universal permission slip for glucose entry: red blood cells and much of the nervous system use uptake mechanisms that are not acutely dependent on insulin. Nor does the liver use the same GLUT4 switch as skeletal muscle. This is why reduced insulin action can change blood glucose dramatically without stopping all glucose uptake. ^[ref]

Diabetes can disrupt production, response, and counterregulation. Type 1 diabetes usually involves destruction of beta cells and severe loss of endogenous insulin; type 2 diabetes combines insulin resistance with insufficient beta cell compensation. High circulating insulin can therefore coexist with impaired insulin action. Inappropriate glucagon release can add to excessive liver glucose output, and the response to low blood glucose can also be impaired: "more glucagon" is not a complete description of diabetes. These distinctions explain mechanisms, rather than prescribe treatment. ^{[ref] [ref]}

A metabotropic receptor couples ligand recognition to intracellular signal transduction, which can regulate a separate ion channel, alter enzyme activity or change gene expression. In the usual neurotransmitter context, metabotropic receptors are G protein-coupled receptors: receptor activation is relayed by a G protein. There need not be a change in membrane potential at all. ^[ref]

Two routes should be distinguished. An activated G protein can regulate an ion channel locally at the cell membrane, without an obligatory diffusible second messenger. Alternatively, it can regulate an enzyme that changes the availability of a second messenger, such as cAMP or inositol trisphosphate. Downstream proteins then change channel opening, phosphorylation or other cellular activities. The receptor, G protein, second messenger and final target are distinct components; an activated G protein is a relay protein, rather than itself a small-molecule second messenger.^[ref]

Do not turn this useful chemical synapse classification into the claim that every receptor is a GPCR, or that every indirect response must pass through a G protein. For example, the insulin receptor is an enzyme-linked cell-surface receptor whose intracellular catalytic activity initiates protein phosphorylation. Intracellular receptors supply another category. “Metabotropic” commonly names the GPCR branch in neuroscience; the broader idea of biochemical coupling does not make all non-channel receptors structurally identical. ^[ref]

Separating recognition from execution allows signal amplification and branching. A persistently activated receptor can activate several relay proteins; a regulated enzyme can produce many second messenger molecules. As an explicitly invented count example, suppose one activated receptor activates four relay proteins during one interval, and each relay activates a distinct enzyme that produces fifteen second messenger molecules during the next interval. With sufficient substrates and no loss, this gives $4\times 15=60$ molecules. Those counts do not specify a physiological gain or response duration. A directly gated ion channel also passes many ions while open, so amplification is not an exclusive property of metabotropic receptors: always name the input and output being compared. ^[ref]

A particularly instructive electrophysiology experiment separated a chemical trigger from the ion channel carrying its eventual electric current. In 1987, Hirono, Ito and Sugiyama injected messenger RNA from rat brain into frog egg cells, then tested acquired responses to neurotensin and muscarinic acetylcholine stimulation. Under voltage clamp at $-60\mathrm{mV}$, neurotensin elicited transient, oscillatory inward electric currents after a delay of several tens of seconds. This delay refers to that response and preparation. Reversal potential measurements implicated increased permeability to chloride anions, rather than a pore specific to the applied ligand. Both neurotensin and acetylcholine responses were inhibited by pertussis toxin, which interferes with susceptible G proteins, and by intracellular EGTA, which binds free calcium. Injecting inositol trisphosphate mimicked the responses; a large prior dose of inositol trisphosphate reduced subsequent responses to either extracellular signal. These interventions implicate a shared intracellular relay and calcium-dependent channel opening.^[ref]

The informative comparison with the nicotinic acetylcholine receptor experiment is mechanistic: an extracellular ligand can produce an inward electric current either by directly gating the pore or by mobilizing an intracellular second messenger and stored calcium that activate another pore. Intracellular inositol trisphosphate bypasses reception, whereas EGTA tests the need for free calcium. However, neither inhibition nor mimicry alone identifies every molecular connection: a perturbation can affect several targets, and an injected mediator can activate a pathway without proving that it is the only natural route. The egg cell's endogenous machinery and the mixed messenger RNA preparation also limit extrapolation to an intact neuron. The experiment establishes functional coupling in its preparation, rather than the exact molecular composition or timing of every metabotropic receptor.

Even G protein-dependent channel regulation need not involve a lengthy second messenger cascade. Pfaffinger and colleagues reported in 1985 that acetylcholine-evoked potassium channel activation in embryonic chick atrial cells required intracellular GTP, a guanine-containing nucleotide, and was abolished by pertussis-toxin pretreatment. Changes in cyclic nucleotides, including cAMP, did not account for the channel response. The report discusses intrinsic activation delays of roughly $30$–$100\mathrm{ms}$, illustrating relatively rapid metabotropic receptor action. These observations support a G protein coupling step without cyclic-nucleotide second messengers; they do not by themselves exclude every possible intermediary or prove the specific subunit-channel interaction.^[ref]

A small timing model makes the extra steps visible without inventing an experimental trace. Let $u(t)$ jump from zero to one at $t=0$ and remain on. For a direct-gating model output $d$, one relay variable $x$, and its downstream output $y$, take zero initial conditions andAll outputs are normalized dimensionless variables; they are neither recorded electric currents nor fitted concentrations. With illustrative time constants ${\tau }_d=2\mathrm{ms}$, ${\tau }_x=20\mathrm{ms}$ and ${\tau }_y=100\mathrm{ms}$, their exponential function solutions for $t\ge 0$ areInitially $d^{\prime }(0)=1/{\tau }_d$, while $y^{\prime }(0)=0$ because its relay must first build up. Both outputs eventually approach one. Equal normalized final values do not imply equal biological amplitudes or absence of signal amplification; normalization deliberately removes that question.

The model's useful feature is the delayed buildup created by sequential relaxation, not its arbitrary numbers. Actual kinetics also depend on ligand delivery, relay deactivation, neurotransmitter clearance and the final target. A local G protein route can act promptly, while an ionotropic receptor can remain active during continued exposure or feed a longer-lasting intracellular response. The beauty of metabotropic receptor coupling is its flexibility: the same recognition event can adjust electrical responsiveness and other cellular tasks through different targets. To classify an observed response, combine timing with molecular identity and controlled perturbations.

Methionine is an amino acid represented by Met or M in protein sequence notation. In the standard genetic code, AUG encodes it, as in the worked gene expression example. ^[ref]

Count the same hypothetical diploid animal biological cell in three ways: chromosomes, double-stranded DNA molecules, and nuclear DNA amount. Assume one continuous double-stranded DNA molecule per unreplicated chromosome or replicated chromatid, complete S phase, and no losses or mutations. Exclude mitochondrion DNA. Here $n=2$ is the size of one haploid set, and $C$ is its unreplicated nuclear DNA amount, as defined in cell-cycle DNA content. A double-stranded DNA molecule counts as one, despite containing two strands.

The allocation can be checked copy by copy. DNA replication turns $A$ into $A_1,A_2$, $a$ into $a_1,a_2$, and similarly for $B,b$. One daughter receives $A_1,a_1,B_1,b_1$ and the other $A_2,a_2,B_2,b_2$. Each therefore inherits the same four chromosome identities and the same gene and allele content as the unreplicated parent, under the stated assumptions. Across both daughters there are $4+4=8$ DNA molecules and $2C+2C=4C$: partition conserves the material present after S phase.

The transient whole-cell count of eight does not mean that each daughter's ploidy has doubled. Each completed daughter is diploid, containing both homologues of each homologous chromosome pair. This differs from separating homologous chromosomes during meiosis I. Faithful mitosis preserves a nuclear genome through growth and renewal; equal inherited genes need not imply equal gene expression or equal cell fate. Mutations or chromosome segregation errors can break the ideal inheritance pattern.

Evidence transfers at a stated level. Sequence alignment can suggest a candidate relationship between genes; functional complementation tests whether a candidate can supply a particular function in a chosen recipient. Neither alone establishes identical native gene expression, identical signal transduction, or identical consequences in every species. The yeast cross-species complementation experiment is especially useful because it tests a conserved function by intervention rather than assuming it from resemblance. ^[ref]

Even successful functional complementation occurs within a specific genetic background, nutrient supply and imposed gene expression. The enzyme may work with the recipient’s available substrates and partner proteins at an experimentally imposed abundance. Establishing the same role in the donor species calls for experiments there; establishing behavior under another condition calls for experiments under that condition. A conserved component can therefore coexist with context-dependent behavior of an entire pathway. This separation makes model organisms powerful: an accessible experiment supplies a precise mechanistic foothold and a better question for the next system.

A morphogen is a signal whose spatial concentration helps specify different biological cell fates or behaviors during biological development. A morphogen gradient provides a positional cue: biological cells at different distances from a source experience different local concentrations and can follow different developmental pathways. A spatial concentration variation alone does not establish that a substance is a morphogen; its graded developmental effects matter. See Alberts et al., Molecular Biology of the Cell, 4th edition (2002): morphogens as graded developmental signals.

The beautiful idea is that one distributed signal can help organize several regions of a developing body. A continuous morphogen gradient can supply the input for distinct domains of gene expression, rather than requiring a separate signal for every position. A morphogen response threshold is a useful idealization of that interpretation: high, intermediate and low concentrations are assigned different outcomes. The morphogen reaction-diffusion equation describes one way to generate a morphogen gradient; the equation is a model of transport and reaction, while the morphogen is the signal being transported.

Detection and interpretation also depend on the receiving biological cell. For an extracellular morphogen, receptors and signal transduction connect the local concentration to a response; receptor binding occupancy is only one step in that connection. The same morphogen gradient can be interpreted differently in different tissues because their developmental context differs. Thus a morphogen response threshold summarizes a particular response in a particular context, rather than being a universal property of the signal. See Scott F. Gilbert, Developmental Biology, 6th edition (2000): positional information and context-dependent interpretation.

An important experimental example came in 1988. Wolfgang Driever and Christiane Nüsslein-Volhard used antibodies to examine the bicoid protein in early fruit-fly embryos. They reported a concentration profile approximately described by an exponential function, highest at the front, with the protein concentrated in cell nuclei. Their abstract proposed diffusion from localized messenger RNA and dispersed degradation as a likely origin of the morphogen gradient, rather than proving a unique transport mechanism. ^[ref]

In a companion study, Driever and Nüsslein-Volhard genetically changed the amount and distribution of bicoid messenger RNA, measured the resulting protein morphogen gradient, and compared developmental maps. Increased local bicoid protein levels shifted prospective anterior structures toward the rear; decreased levels shifted them toward the front. This linked a measured concentration profile to positional effects in biological development. The toy calculations below illustrate that logic with arbitrary numbers, not measured bicoid parameters. ^[ref]

A morphogen response threshold is a concentration level used to distinguish developmental responses to a morphogen. In an idealized threshold model, otherwise equivalent biological cells exposed to $c\ge \theta$ take one response and those exposed to $c<\theta$ take another. Two morphogen response thresholds can assign three bands: fate A for $c\ge 50$, fate B for $20\le c<50$, and fate C for $c<20$, all in the toy arbitrary concentration units. The A/B/C labels specify a response rule, not named cell types or a claim that every morphogen produces sharp boundaries. See the positional-information model in Gilbert's reference under morphogen.

For the decreasing morphogen gradient $c(x)=c_0{e}^{-x/\ell }$ and $0<\theta <c_0$, solve $c(x_{\theta })=\theta$ using the natural logarithm:With $c_0=100$ and diffusion length $\ell =100\mu \mathrm{m}$, the morphogen response thresholds $50$ and $20$ giveThus fate A occupies $0\le x\le x_{50}$, fate B occupies $x_{50}<x\le x_{20}$, and fate C occupies $x>x_{20}$ in this idealization. The equality convention belongs to the response rule. If $\theta =c_0$, the crossing is at the source; if $\theta >c_0$, that morphogen response threshold is never reached anywhere in the tissue.

Now double the prescribed source concentration from $c_0$ to $2c_0$, keeping $D$, $k$, diffusion length $\ell$ and a fixed morphogen response threshold $0<\theta <c_0$ unchanged. The whole morphogen gradient doubles, but the boundary moves by an additive distance:For $\theta =20$, the crossing moves from $160.9438\mu \mathrm{m}$ to $100\ln 10\mu \mathrm{m}\approx 230.2585\mu \mathrm{m}$, a shift of $69.3147\mu \mathrm{m}$. Every fixed morphogen response threshold below the original source concentration shifts by this same distance in the toy model. The example compares two Dirichlet boundary conditions, $c(0)=100$ and $c(0)=200$; doubling an experimentally supplied source flux is a different boundary specification.

These calculations are useful because they separate changes in source concentration, diffusion length and response rule. Their limits are equally concrete: growing tissues change the geometry, a transient response can precede steady state, molecular fluctuations can blur a boundary, and signaling feedback or altered transport can change the morphogen gradient or its interpretation. A local instantaneous morphogen response threshold need not capture exposure duration, signaling adaptation or the receiving biological cell's state. Experiments on cultured human biological cells found different responses to the level and rate of change of developmental signals, illustrating why signal dynamics can matter. ^[ref] Quantitative measurements of bicoid protein and its downstream gene expression also make precision a question for experiment, rather than something guaranteed by drawing threshold lines. ^[ref] Morphogen gradients provide one mechanism for spatial patterning in biological development, alongside other signals and interactions.

A multicellular organism has a body of multiple biological cells whose growth and activities are integrated into the life of the whole organism. Extensive cell differentiation is common in complex examples, but specialized biological cells are not a prerequisite for every simple form. Ask how the body develops, how its parts cooperate and how it produces descendants, rather than counting attached biological cells alone. The boundary with a biological colony depends on the degree of integration and the criteria being investigated; there is no universal cell-count threshold. See the Queller and Strassmann account cited under unicellular organism.

Specialization makes the whole capable of combinations that one general-purpose biological cell cannot achieve in the same way. In an animal, a neuron transmits signals while a red blood cell carries oxygen; each still requires a working cell membrane and metabolism. A mature mammalian red blood cell lacks a cell nucleus and mitochondria, illustrating how a useful part need not retain the equipment of a self-reproducing organism. See Clark, Douglas and Choi, Biology 2e, 40.2: red blood cell specialization. Cell differentiation generally involves different gene expression, rather than a different inherited genome for each function. Biological development must coordinate growth, position, interactions and specialization: increasing the number of biological cells is only one component. See Alberts et al., Molecular Biology of the Cell, chapter 21 (2002): construction of multicellular bodies.

A motivating experiment: select for a property of groups. Ratcliff, Denison, Borrello and Travisano's 2012 study began with Saccharomyces cerevisiae growing as unicellular organisms. Daily transfer favored rapidly settling material. Their initial protocol allowed a $10\mathrm{mL}$ culture to settle for $45\min$, discarded the upper $9.9\mathrm{mL}$, and transferred the bottom $100\mu \mathrm{L}$ into fresh medium. After seven days, they instead sampled $1.5\mathrm{mL}$ and centrifuged it at $100$ times gravitational acceleration for $10\mathrm{s}$, again transferring the bottom $100\mu \mathrm{L}$. Faster settling made it more likely that a lineage would seed tomorrow's culture; the procedure selected a physical outcome without specifying how it must arise. These protocol details are explicitly recounted in Gulli, Herron and Ratcliff, Evolution of altruistic cooperation among nascent multicellular organisms (2019): original settling protocol.

The primary 2012 paper reports snowflake yeast dominating all ten initial replicate populations by 60 transfers. To distinguish the joining of previously separate biological cells from clonal development, the researchers enzymatically separated clusters and followed individual biological cells for $16\mathrm{h}$: attached descendants rebuilt clusters, with no such joining observed. Staining localized attachments at bud scars. Time-lapse observations also showed clusters releasing smaller multicellular offspring, called propagules; small clusters grew before they reproduced. See Ratcliff et al., Experimental evolution of multicellularity (2012), figures 1–3: emergence, development and reproduction.

The experimental comparisons ask different questions. Competition with the unicellular organism ancestor showed a reported $34\%$ advantage under settling selection. Without settling selection, the estimated $10\%$ disadvantage had a one-sided $P=0.06$, so it was suggestive rather than decisive evidence of a cost. Separately, three populations derived from one evolved isolate retained the cluster form through 35 transfers without settling selection. That persistence tests stability, not whether clusters always outperform single biological cells. See Ratcliff et al. (2012), figure 2 and the cluster-formation results cited above.

A 2015 follow-up added a causal test: deleting the gene ACE2 in the unicellular organism ancestor produced snowflake yeast, while supplying functional ACE2 to one evolved isolate restored separation into single biological cells. Sequence changes altering the encoded protein were found in ACE2 in five of ten examined lineages; the study did not identify that same sequence-change mechanism in every lineage. The deletion and restoration comparisons support a mechanism beyond the observation that clusters settle quickly. See Ratcliff, Fankhauser, Rogers, Greig and Travisano, Origins of multicellular evolvability in snowflake yeast (2015), figure 1.

What follows, and what does not. The evidence supports inherited cluster formation, clonal development and a reproducing collective whose properties can respond to natural selection. Calling these simple multicellular organisms recognizes those demonstrated properties; it does not turn every clump into an animal or establish animal-like cell differentiation. This laboratory route also does not reconstruct the actual ancestry of animals or show that complex developmental integration must follow. The interesting achievement is narrower and testable: a change in how descendants remain connected creates a new scale at which growth and reproductive success can be measured.

For the toy figure, let $p$ be the fraction of tracked units of type C before transfer and let $s_C,s_U$ be the respective probabilities of transfer for C and U. Assume equally weighted units and equal multiplication between transfers. When overall transfer probability is nonzero, conditioning on transfer givesWith $p=1/5$, $s_C=4/5$ and $s_U=1/10$, the numerator is $4/25$ and the denominator $6/25$, giving $p_{\mathrm{after}}=2/3$. If both types have the same nonzero transfer probability, the fraction stays $1/5$. This is conditional probability, not a model of the full experimental life cycle: real cluster sizes, growth and fragmentation affect which quantity a frequency measures. In particular, a fraction of clusters is not automatically a fraction of biological cells.

The N-terminus is the amino end of a protein or amino acid chain. Sequence notation lists residues from the N-terminus toward the C-terminus, the same direction in which biological translation builds a chain. The two-residue example under gene expression places methionine at the N-terminal end before any subsequent processing. ^[ref]

A polypeptide is a chain of amino acid residues linked by peptide bonds. The term describes chain chemistry; calling a chain a polypeptide alone does not establish that it is folded or functional. The gene expression example N–methionine–alanine–C contains two amino acid residues and one peptide bond; the twelve-residue example under protein contains eleven peptide bonds. See IUPAC-IUB: amino-acid symbols and representation of residues in chains.

A gene promoter is a DNA region involved in initiating transcription. Moving a coding sequence into a nuclear genome does not guarantee that its original gene promoter can drive nuclear gene expression.

A biochemical receptor is a protein that recognizes a signal and couples its detection to a biological effect. Its binding partner is a ligand; recognition depends on compatible shape and chemical interactions at a binding site within the protein structure. Specificity is selective recognition, not a guarantee that only one ligand can bind. Binding also need not activate the receptor: a competing ligand may occupy it while preventing activation by another ligand. See Alberts et al., Molecular Biology of the Cell, 4th edition: protein binding and specificity.

Compare a cell-surface receptor, accessible to an extracellular ligand, with an intracellular receptor, whose ligand must reach the interior. A cell-surface receptor can detect a hydrophilic ligand without transporting it through the cell membrane. In contrast, some membrane-permeable signals reach intracellular receptors that regulate gene expression. Location and downstream coupling are separate distinctions: an ionotropic receptor directly gates an ion channel, whereas a metabotropic receptor works through intracellular signal transduction. See Clark, Douglas and Choi, Biology 2e, 9.1: receptor locations and mechanisms.

Receptor binding occupancy quantifies recognition separately from response. A dissociation constant measures binding in a specified equilibrium model; it does not by itself predict the cellular effect. This separation is useful when comparing signals that bind equally well but produce different responses, or when interpreting a binding experiment alongside a functional measurement.

Signal transduction converts a detected signal into a change inside a biological cell. For chemical signals, distinguish reception by a receptor, intracellular signal transduction, and the response: altered gene expression, enzyme activity or ion channel opening, for example. A cell-surface receptor can relay a ligand's presence across a cell membrane without the ligand entering the cytoplasm. Thus a hydrophilic signal need not cross the membrane to influence the interior. See Clark, Douglas and Choi, Biology 2e, 9.2: intracellular signal propagation.

For a computed example of possible signal amplification, suppose one activated receptor activates five relay proteins, and each relay activates twenty target proteins during a chosen interval. If the targets are distinct and available, $1\times 5\times 20=100$ targets are activated. Those assumed counts illustrate multiplication, not a measured pathway or a universal gain. Actual signal transduction can branch, saturate or inhibit activity. Changes in gene expression and changes in existing enzyme activity are different responses. See Clark, Douglas and Choi, Biology 2e, 9.3: cellular responses.

Signal amplification, signaling feedback and signaling adaptation are possible properties of signal transduction, rather than requirements of every cascade. Intracellular relays can use proteins or second messengers; different biological cells can interpret the same ligand differently because they contain different receptors and relay machinery. The useful and striking idea is that recognition and response are separable: an extracellular chemical message can control many internal tasks without itself performing those tasks. See General Principles of Cell Communication.

An original experimental route to this separation appears in T. W. Rall and Earl W. Sutherland's 1958 work on cAMP. They described a heat-stable factor formed from ATP in separated biological cell preparations; it affected enzyme activity in other preparations where the initiating chemical signals had no direct effect. This helped distinguish an initial signal from a molecule carrying its effect to another target. ^[ref]