November 15, 19784
Dear Dan:

I have almost completed the sequencing of the mPRL, mGH, and mPLC cDNA
clones, but I am now at the point that I can predict the complete amina
acid sequence for each protein. A few ambiguities remain in two of the
sequences in the 3’-untranslated region and part of these sequences have
been determined on only one strand (due to the limitations of reading
through the G-C tails). I need to verify the mPRL sequence because of
two odd findings. First, the published protein sequence for mPRL begins
with Leu-Pro-Ile-etc. The cDNA sequence predicts that the amino acid at
the —-1 position is a proline, and no protein reported in Von Hei jne‘s
review paper cantains a proline residue at this position. Second, the
initiator ATG for mFPRL, based on hamology to other prolactins and on the
length of the leader sequence, is just downstream of another ATG, and
this other ATG. appears to obey the rules for a strong translation start
site. If I can verify these findings by sequencing independent mPRL cDNA
Clones, they may add some twists to the signal sequence cleavage and
translation initiation dogmas.

Io am enclosing the amino acid sequences for mPRL, mGH, and mPLC (the
Placental cDNA clone that hybridizes weakly to proliferin). They are
given with the homology of mPLC to each of mPRL, mGH, and mPLF. mPLC has
several interesting features: (1) as predicted from the larger mRNA, mPLC
encodes a protein larger than any of the other three —- 244 amino acids in
the precursor; (23) mPLC lacks the amino terminal two Cys residues (the
same as mGH) and has the remaining 4 Cys residues common to the other
three proteins, but it has an extra Cys as well (circled in green); (3)
the protein extends beyond the final Cys, again similar to GH: (4) this
stranger structural homolagy to GH is not repeated in comparisons of
sequence homology; (5) the first 10 amino acids are identical to those
predicted from the sequence of PLF-1 (this is extremely surprising since
the Leu at position 3S in PLF-1i is predicted to be a Ser from PLF-? and
from Krebs RNA); (4) this region of homology is reflected in the
nucleotide sequence - see the other enclosure — where mPLF and mPLC are
nearly identical throughout the S‘-untranslated region and into the
coding region (this hamology is suggestive of an exon shuffling event in
the not too distant past; I do nat know the exon boundary, but my quess
is that it will come between the codons for amino acids i190 and 11;
undoubtedly, this region explains the cross-hybridization); (7) certain
regions in mPLC are more homologous to bPRL than to mPRL, suggesting that
mPLO and mPRL have drifted apart in those regions following gene
duplication faster than a mouse and a bovine gene have drifted apart from
a common gene in some mammalian ancestor; for example:

92 135
mPLC ITEAFNSCHT FLEHLVTE
mPRL MVE VIN DCP T PLFQLITG
BPRL TFMALNSCHT FLYHLVTE

(8) mPLC has 3 potential glycosylation sites, all in the amino terminal

half of the protein, and 2 Arg-Lys sequences. I am sure that you will
find some other points of interest that I have neglected. I have not yet

put together the sequence comparisons between the mammalian GH genes and
between the mammalian PRL genes; that will be done in the near future.
T am curious to hear how the proliferin story is developing outside of
Tllinois. Although I am quite busy here and am enjoying my interactions
with the other labs in the department, I do feel a bit out of touch.
Finally, TI have another favor toa ask of you. I have applied for a Basil
O’°Conner Starter Grant from the March of Dimes, and they would like a
letter from you describing my potential to become an independent
scientist. Would you be willing to write an my behalf to

Samuel J. Ajl, PH.D.

Vice Fresident for Research

Basil O’Conner Starter Research Grant

March of Dimes Birth Defects Foundation

1275 Mamaroneck Avenue

White Flains, New York 10405
The application and supporting letters are due by January 1, 19985. Thank
you in advance.

Sincerely,

7.5. 1) Did you hke cave of the espe t Plailpsean ov stale I?
2) Whun L uviteup fle PLC Sejuence, do we uant fe
3) LD pad wilser- Homiltms Peges fr barns & Phe
Die Sein — 33 aowythens hagyering on thatfout .

\