Variable and constant regions are separated in the 10-kbase transcription unit coding for immunoglobulin kappa light chains.

UV transcription mapping with recombinant DNA probes containing immunoglobulin kappa light chain mRNA sequences has been used to determine the size of the transcription unit coding for kappa light chain m RNA and to establish the arrangement of variable and constant regions in this transcription unit. In relation to ribosomal RNA standards, the transcription of kappa light chain constant region sequences into nuclear RNA exhibits a UV target size of 9.6 kbases (kb). The kappa light chain variable region exhibits a UV target size of 7.6 kb indicating that it is separated by approximately 2.0 kb from the constant region in the kappa light chain transcription unit. The size of the primary transcript (i.e., the direct, unprocessed RNA product of transcription) predicted from the constant region target size concurs with our previous pulse-labeling results which showed that the largest presumptive nuclear RNA precursor to kappa light chain mRNA is approximately 10 kb. In addition, the UV target size of cytoplasmic kappa mRNA is indistinguishable from the target size of constant region sequences in nuclear RNA. These results suggest that the kappa light chain transcription unit is copied directly into a 10-kb nuclear RNA precursor in which the kappa variable and constant regions are separated by approximately 2 kb. Accordingly, it is proposed that the joining of immunoglobulin kappa light chain variable and constant regions occurs in the post-transcriptional processing of this large nuclear RNA precursor into kappa light chain mRNA.

Utilization of early promotors in mutant far P85 of bacteriophage T4.

We show that farP85 is a recessive mutant of T4 incapable of activating the delayed early promotors for genes 43 and 45 and that the farP85 mutation is in the same complementation group as the ts G1 mutation, which is located in the "modifier of transcription" (mot) gene.

Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's.

In vivo decay rates for the individual T7 early mRNA species were determined. The physical half-lives, measured at 37 C, range from 1.1 min for gene 0.7 RNA to 4.5 min for gene 0.3 RNA. Physical half-lives, as observed after rifampin inhibition of RNA synthesis and polyacylamide electrophoresis of RNAs, are approximately 30% longer than functional half-lives, as observed by 14C-labeled amino acid uptake into individual T7 early proteins. The different RNA species are synthesized at grossly different rates, 0.3 RNA at four times the rate of 1.0 RNA, 0.7 RNA at twice the rate, and 1.1 and 1.3 RNAs at about the same or a slightly lower rate than 1.0 RNA. Rho-factor-mediated termination of transcription behind genes 0.3, 0.7, and perhaps behind 1.0 is inferred from these data. The in vivo translational utilization of the individual T7 early-message species was found to vary by not more than a factor of 2.

Two modes of in vivo transcription for genes 43 and 45 of phage T4.

Sensitivities of the expression of early genes of phage T4 to UV light were determined at various stages of intracellular development of T4 wild type, a DNA-negative mutant (T4 DO), and T4 tsG1, (a mutant that exhibits delayed expression of some T4 early genes). Whereas the sensitivities of some genes in the T4 wild type and T4 DO remain constant, genes 43 and 45 exhibit greatly reduced sensitivities several minutes after the onset of phage development. Since UV sensitivities are a measure of the distance of a gene from its promotor, these observations indicate a switch from distal, "immediate early" promotors to proximal, "delayed early" promotors for genes 43 and 45. In the tsG1 mutant this decrease in UV sensitivities of genes 43 and 45 does not occur at 42 C, suggesting that at high temperature this mutant does not utilize the delayed early promotors.

Cleavage by RNase 3 converts T3 and T7 early precursor RNA into translatable message.

The processing of the early T3 and T7 high-molecular-weight precursor messenger RNA by RNase III is necessary for its efficient translation both in vivo and in vitro.

SP62, a viable mutant of bacteriophage T4D defective in regulation of phage enzyme synthesis.

SP62 is a mutant of bacteriophage T4D that was discovered because it produces fewer phage than the wild type in the presence of 5-fluorodeoxyuridine. In the absence of phage DNA synthesis, SP62 solubilizes host DNA slower than normal; this may explain the sensitivity to 5-fluorodeoxyuridine. In Escherichia coli B at 37 C in the absence of drugs, SP62 makes DNA at a normal rate and the kinetics of appearance of phage are nearly normal. Under the same conditions, SP62 produces T4 lysozyme (gene e) at a normal rate until 20 min, but then produces it at twice the normal rate until at least 60 min. It has long been known that, when T4 DNA synthesis is blocked (DNA(-) state) in an otherwise normal infection, the synthesis of a number of early enzymes continues beyond the shutoff time of about 12 min seen in the DNA(+) state, but still stops at about 20 min. We have termed the 12-min shutoff event S1 and the 20-min shutoff event S2. We show here that, in the DNA(+) state, SP62 makes four early enzymes normally, i.e., S1 occurs. However, in the DNA(-) state (where S1 is missing), SP62 continues to make dCTPase (gene 56), dCMP hydroxymethylase (gene 42), and deoxynucleotide kinase (gene 1) for at least an hour; this results in production of up to 13 times the normal level of dCTPase at 60 min after infection, or 6 times the DNA(-) level. We conclude that SP62 is defective in the second shutoff mechanism, S2, for these three enzymes. In contrast, SP62 causes premature cessation of dTMP synthetase production in the DNA(-) state; the result is a twofold underproduction of dTMP synthetase. Autoradiograms of pulse-labeled proteins separated by slab-gel electrophoresis in the presence of sodium dodecyl sulfate show that a number of other T4 early proteins, including the products of genes 45, 46, and rIIA, are synthesized longer than normal by SP62 in the DNA(-) state. Few late proteins are made in the DNA(-) state, but in autoradiograms examining the DNA(+) state there is little or no effect of the SP62 mutation on the synthesis of T4 late or early proteins. Circumstantial evidence is presented favoring a role for the gene of SP62 in translation of certain mRNAs. At very high temperatures (above 43 C) in the absence of drugs, phage production, but not DNA synthesis, is much reduced in SP62 infections relative to wild-type T4 infections; this temperature sensitivity is greater on E. coli CR63 than on E. coli B. This property has facilitated recognition of the SP62 genotype and aided in complementation testing and genetic mapping. A later publication will provide evidence that SP62 defines a new T4 gene named regA, which maps between genes 43 and 62.

Transcription units in bacteriophage T4.

We have investigated the possibility of assigning genes of T4 bacteriophage to their units of transcription (scriptons) by studying gene expression from UV-irradiated DNA templates. Since RNA chains are prematurely terminated on UV-irradiated DNA templates and since the promotor distal part of the RNA chain is deleted, the expression of any gene is inversely proportional to the distance between the promotor and the promotor distal end of the gene. We find that the early genes, 43, 45 and rIIB, are promotor proximal. Since at least genes 43 and rIIB are classified as delayed early genes, these results suggest that their synthesis may require the recognition of new promotors. Additional early genes (44, 62, 42, 46, 47, 55, and rIIA) and some late genes (34, 37, and 38) have also been assigned positions relative to their promotors.

Control of gene function in bacteriophage T4. IV. Post-transcriptional shutoff of expression of early genes.

The selective and sequential shutoff of synthesis of early T4 proteins in bacteria infected with DNA-negative mutants is under the active control of one or more T4-induced proteins. Selective shutoff of synthesis of early T4 proteins is accompanied by a selective degradation of distinct species of T4 mRNA. We present circumstantial evidence that selective degradation of mRNA is the cause, and not the consequence, of selective termination of expression of early T4 genes. The mutation sp62 inactivates the shutoff mechanism and prevents the selective degradation of distinct species of T4 mRNA.

Specific suppression of mutations in genes 46 and 47 by das, a new class of mutations in bacteriophage T4D.

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.

Mutants in a nonessential gene of bacteriophage T4 which are defective in the degradation of Escherichia coli deoxyribonucleic acid.

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.