Nd:YAG laser machining with multilevel resist kinoforms.

We demonstrate that resist kinoforms can be used for laser micromachining. A 10-level resist kinoform, manufactured by electron-beam lithography, was shown to have a diffraction efficiency of 68%. Nine diffraction-limited holes were simultaneously drilled in 0.10-mm-thick stainless steel. Marking in a silicon wafer is also demonstrated.

Partially illuminated kinoforms: a computer study.

The properties of kinoforms being fully and partially illuminated during synthesis are compared by computer simulation. Partial illumination during synthesis results in smoother intensity patterns produced by the kinoforms. A new iteration technique which uses a Gaussian beam that moves during kinoform synthesis is proposed.

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells.

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.

In vivo expression of the rII region of bacteriophage T4 present in chimeric plasmids.

The expression of the T4 rII genes in uninfected cells has been examined by use of recombinant plasmids. Hybridization analysis of pulse-labelled RNA prepared from cells carrying pTB101, a plasmid that contains the end of T4 gene 60 and the beginning of gene rIIA, shows that about 0.7% of the labelled RNA is rII specific. By contrast, only 0.02% of pulse-labelled RNA prepared from cells carrying plasmid pTB301, which probably contains the middle-mode rIB promoter, may be rII specific. When separated strands of T4 DNA were used for hybridization, we found that the pTB101 transcripts have a strand specificity identical to that of the rIIA transcripts made during phage infection. The same strand specificity was observed irrespective of the orientation of the inserted DNA in the vector. This result argues that the transcripts initiate within the inserted DNA rather than somewhere else on the plasmid. We also found that essentially none of the pulse-labelled pTB101 RNA would hybridize to the DNA of a T4 deletion mutant that lacks the rIIA gene. This suggests that little of the gene 60 DNA of the plasmid is being transcribed. In addition to the rII transcript, a new protein of 56,000 Daltons molecular weight is found in cells carrying pTB101. Fingerprint analysis of the protein shows that it is specified by the rIIA gene of the plasmid. Taken together, these results indicate that transcription of the plasmid rIIA gene initiates at or near the beginning of the gene.

Construction and properties of recombinant plasmids containing the rII genes of bacteriophage T4.

The EcoRI digestion products of phage T4 DNA have been examined using a phage DNA transformation assay. A 2.6 X 10(6) Dalton fragment was found to contain the rII genes. This fragment was purified and then treated with HindIII endonuclease. The cleavage products were ligated to the vector plasmid pBR313 and viable recombinant plasmids recovered. A genetic assay was employed to demonstrate that the recombinants contained T4 DNA and to localize on the phage genetic map the EcoRI and HindIII sites cleaved during the construction of the plasmids. Preliminary characterization suggests that a fragment covering the beginning of the rIIA gene possibly contains a promotor which is active in uninfected cells.

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes.

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56, denA and denB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA with EcoRI have been cloned using the vector plasmid pCR1. Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4.